(Issues in Toxicology No. 26) Jenner, John - Thiermann, Horst - Worek, Franz-Chemical Warfare Toxicology, Volume 1-Royal Society of Chemistry (2016)

Chemical Warfare Toxicology
Volume 1: Fundamental Aspects

Issues in Toxicology
Series Editors:
Professor Diana Anderson, University of Bradford, UK
Dr Michael D. Waters, Michael Waters Consulting, N. Carolina, USA
Dr Timothy C. Marrs, Edentox Associates, Kent, UK
Advisor to the Board:

Dr Alok Dhawan, Ahmedabad University, India
Titles in the Series:

1: Hair in Toxicology: An Important Bio-Monitor
2: Male-mediated Developmental Toxicity
3: Cytochrome P450: Role in the Metabolism and Toxicity of Drugs and
other Xenobiotics
4: Bile Acids: Toxicology and Bioactivity
5: The Comet Assay in Toxicology
6: Silver in Healthcare
7: In Silico Toxicology: Principles and Applications
8: Environmental Cardiology
9: Biomarkers and Human Biomonitoring, Volume 1: Ongoing Programs
and Exposures
10: Biomarkers and Human Biomonitoring, Volume 2: Selected Biomarkers
of Current Interest
11: Hormone-Disruptive Chemical Contaminants in Food
12: Mammalian Toxicology of Insecticides
13: The Cellular Response to the Genotoxic Insult: The Question of
Threshold for Genotoxic Carcinogens
14: Toxicological Effects of Veterinary Medicinal Products in Humans:
Volume 1
15: Toxicological Effects of Veterinary Medicinal Products in Humans:
Volume 2
16: Aging and Vulnerability to Environmental Chemicals: Age-related
Disorders and their Origins in Environmental Exposures
17: Chemical Toxicity Prediction: Category Formation and Read-Across
18: The Carcinogenicity of Metals: Human Risk Through Occupational and
Environmental Exposure
19: Reducing, Refining and Replacing the Use of Animals in Toxicity Testing
20: Advances in Dermatological Sciences
21: Metabolic Profiling: Disease and Xenobiotics
22: Manganese in Health and Disease
23: Toxicology, Survival and Health Hazards of Combustion Products
24: Masked Mycotoxins in Food: Formation, Occurrence and Toxicological
Relevance
25: Aerobiology: The Toxicology of Airborne Pathogens and Toxins
26: Chemical Warfare Toxicology, Volume 1: Fundamental Aspects
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Chemical Warfare Toxicology
Edited by
Franz Worek
Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
Email: franzworek@bundeswehr.org
John Jenner
Defence Science and Technology Laboratory, Porton Down, UK
E-mail: jjenner@dstl.gov.uk
Horst Thiermann
Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany
E-mail: horstthiermann@bundeswehr.org
Issues in Toxicology No. 26
Print ISBN: 978-1-84973-969-6

Two-volume set print ISBN: 978-1-78262-804-0
PDF eISBN: 978-1-78262-241-3
EPUB eISBN: 978-1-78262-806-4
ISSN: 1757-7179
A catalogue record for this book is available from the British Library
© The Royal Society of Chemistry 2016
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Preface
The international community has undertaken great efforts to abandon

chemical weapons. This endeavour resulted in an agreement, the Chemical
Weapons Convention, which entered into force on 29th April 1997 and has
been ratified and implemented by, at present, 192 state parties. This conven-
tion bans the production, stockpiling and use of chemical weapons, and up
to now more than 90% of the declared stockpiles have been destroyed.
Despite this great success, recent events have demonstrated the continuing
threat of chemical warfare agents. The repeated homicidal use of the chemi-
cal warfare nerve agent sarin in Syria in 2013, the suspected perpetual use of
chlorine in Syria and most recently supposed attacks with sulphur mustard
in Syria and Iraq illustrate the capacity and intent to deploy chemical warfare
agents against military units and civilians.
The decreasing likelihood of state use of chemical weapons and increasing
interest of terrorist groups in using toxic chemicals as weapons will have a
great impact on the spectrum of future threat agents, modes of dissemina-
tion and, consequently, preparation of countermeasures for military forces
and the general population.
The intention of this book, Chemical Warfare Toxicology is to provide an
overview of the toxicological properties of relevant chemical warfare agents
including nerve, blistering and lung agents, and opioids. The main focus of
this multi-author book is to give an update on recent findings for a broad
range of topics related to chemical warfare agents. This includes chapters
on new insight into the toxicology and pathophysiology of nerve, blistering
and lung agents, established and experimental means for diagnosis and ver-
ification of exposure to chemical warfare agents, novel approaches for the
treatment of nerve agent poisoning, and an update on the use of, and human
exposure to, chemical warfare agents.

Chemical Warfare Toxicology, Volume 1: Fundamental Aspects
Edited by Franz Worek, John Jenner, and Horst Thiermann
Published by the Royal Society of Chemistry, www.rsc.org
vii
viii Preface
The chapters of this book are authored by experts covering the broad range
of topics related to chemical warfare agents. The authors are regarded as
authorities in the fields of toxicology and military medicine, presenting state
of the art information for academic, clinical and governmental audiences.
The first volume, Fundamental Aspects, covers the fundamentals of the tox-
icology of nerve agents and vesicants whilst the second volume, Management
of Poisoning, describes aspects of the treatment after exposure to these and
other chemical warfare agents.
References to Material in the
National Archives
Over the years, many of the reports produced as accounts of Government

research have found their way into the public records of the originating
nation. In the USA the military reports relating to Chemical Warfare Agents
that are available for public review are indexed by the Defense Technical
Information Center (DTIC) and can be accessed via the website http://www.
dtic.mil. In the UK, similar reports are held in The National Archives and can
be accessed through the website http://www.nationalarchives.gov.uk. In this
book, the references to reports in The National Archives (UK) are referenced
using the system used by the archive, so that each report can be searched for
and located using its unique reference number. The number consists of a
two letter code identifying the contributing department and a series number.
The series number may consist of two numbers separated by a forward slash.
A piece may be a single item or a collection of documents. If the reference is
part of a collection, an additional item number may be added to identify the
document within the piece. For example, WO 1234/56/7 would indicate item
7 of piece 1234/56 entered into the archive by the War Office.

ix

Contents
Chapter 1 Development, Historical Use and Properties of

Chemical Warfare Agents 1
Robin Black
1.1 Introduction 1
1.2 Brief History of CW 2
1.2.1 Prior to 1914 2
1.2.2 The 1914–18 War (WWI) 2
1.2.3 The Inter-War Years 4
1.2.4 The 1939–1945 War (WWII) 5
1.2.5 Post WWII and the Cold War Years 6
1.2.6 The Middle East 7
1.2.7 Terrorism 8
1.2.8 Chemical Weapons Convention 8
1.3 Classification, Properties and Modes of Use of CW Agents 9
1.3.1 Classification 9
1.3.2 Physicochemical Properties 9
1.3.3 Ease of Production 12
1.4 Main Classes of Chemical Agents 12
1.4.1 Lung Injurants (Choking Agents) 12
1.4.2 Blood Agents 13
1.4.3 Vesicants (Blister Agents) 14
1.4.4 Nerve Agents 16
1.4.5 Riot Control Agents 19
1.4.6 Incapacitants 20
1.4.7 Future Developments 22
References 23

xi
xii Contents
Chapter 2 Toxicology of Vesicants 29
John Jenner
2.1 Introduction 29
2.2 Sulfur Mustard 30
2.2.1 Mechanism of Action 30
2.2.2 Toxicokinetics, Metabolism and Distribution 32
2.2.3 Acute Toxicity 36
2.2.4 Irritation and Corrosiveness 44
2.2.5 Sensitisation 48
2.2.6 Repeated Dose Toxicity 49
2.2.7 Mutagenicity 52
2.2.8 Carcinogenicity 54
2.2.9 Toxicity for Reproduction 58
2.2.10 Summary of SM Toxicology 60
2.3 Lewisite 60
2.3.1 Toxicokinetics 61
2.3.2 Acute Toxicity 62
2.3.3 Ocular Toxicity 65
2.3.4 Repeated Dose Toxicity 66
2.3.5 Mutagenicity 67
2.3.6 Toxicity for Reproduction 67
2.3.7 Summary of L Toxicology 68
2.4 Conclusions 68
Acknowledgements 69
References 69
Chapter 3 Toxicology of Organophosphorus Nerve Agents 81

Helen Rice
3.1 Introduction 81
3.2 General Substance Information 82
3.2.1 Physicochemical Properties 82
3.2.2 History 83
3.2.3 Uses 86
3.3 Hazard Characterization of Nerve Agents 86
3.3.1 Acute Effects of Nerve Agent Exposure 87
3.3.2 Historical Nerve Agent Toxicity Studies 88
3.3.3 Other Effects of Nerve Agent Exposure 100
3.3.4 Delayed and Long Term Effects of Nerve
Agent Exposure 101
3.3.5 Effects of Low Level Nerve Agent
Exposure 102
3.4 Human Estimates of Nerve Agent Toxicity 104
3.5 Summary 104
References 107
Contents xiii
Chapter 4 Toxicology and Treatment of Phosgene Induced
Lung Injury 117
Bronwen Jugg
4.1 Introduction 117

4.2 Properties 119
4.2.1 Odour 119
4.2.2 Pathophysiology 119
4.3 History of Use 123
4.3.1 Warfare 123
4.3.2 Occupational/Accidental Exposures 124
4.4 Haber’s Law 125
4.5 Tolerance 127
4.6 Mechanisms of Phosgene Injury 128
4.7 Therapeutic Research Approaches 130
4.7.1 In vitro Studies 131
4.7.2 Small Animal In vivo Studies 134
4.7.3 Large Animal In vivo Studies 136
4.8 Recent Advances 141
4.8.1 Potential Future Therapeutic Options 143
4.9 Conclusions 146
Acknowledgements 147
References 147
Chapter 5 Human Exposures to Sulfur Mustard 154

John Jenner

5.2 Toxic Effects of SM in Humans 155
5.2.1 Effects on the Eyes 155
5.2.2 Effects on the Skin 158
5.3 Conclusions 173
References 175
Chapter 6 Long-Term Effects of the Chemical Warfare Agent

Sulfur Mustard 179
Kai Kehe, Dirk Steinritz, Frank Balszuweit,
and Horst Thiermann

6.2 Sulfur Mustard 180
6.2.1 Cutaneous Injury 181
6.2.2 Ocular Injury 183
6.2.3 Pulmonary Injury 185
6.2.4 Cancers 186
References 187
xiv Contents
Chapter 7 Toxicokinetics of Sulfur Mustard 191
Jan P. Langenberg, Marcel J. van der Schans,
and Daan Noort

7.2 Experimental 193
7.2.1 Analytical Procedures 193
7.2.2 Animal Models 194
7.3 Toxicokinetics of Sulfur Mustard 195
7.3.1 Intravenous Toxicokinetics of Sulfur
Mustard 195
7.3.2 Subcutaneous Toxicokinetics of Sulfur
Mustard 200
7.3.3 Inhalation Toxicokinetics of Sulfur
Mustard 201
7.3.4 Percutaneous Toxicokinetics of Sulfur
Mustard 204
7.4 The Influence of Scavengers on the Toxicokinetics
of Sulfur Mustard 206
7.5 Toxicokinetics of Sulfur Mustard in Humans 207
7.6 Conclusions 209
References 210
Chapter 8 Modeling Organophosphorus Chemical Warfare

Nerve Agents: A Physiologically Based
Pharmacokinetic–Pharmacodynamic (PBPK-PD)
Model of VX 213
Tammie R. Covington, Lucille A. Lumley,
Christopher D. Ruark, Edward D. Clarkson,
Christopher E. Whalley , and Jeffery M. Gearhart

8.2 Materials and Methods 215
8.2.1 PBPK Model Structure 215
8.2.2 Model Parameters 218
8.2.3 Data Used for Model Parameterization
and Validation 222
8.2.4 Sensitivity Analyses 223
8.3 Results 224
8.3.1 Simulations 224
8.4 Discussion 231
8.4.1 PBPK Model Structure 231
8.4.2 Model Parameters 251
8.4.3 Simulations 253
Contents xv
8.5 Conclusions 258
Acknowledgements 258
References 259
Chapter 9 Allometric Modeling of Mammalian Cyanogen

Chloride Inhalation Lethality 264
Douglas R. Sommerville

9.2 Statistical Background 265
9.2.1 Allometric Modeling and IH Toxicology 266
9.2.2 IH Dose–Response Statistics and the
Toxic Load 268
9.2.3 Probit Analysis Models Used for Fitting
Response Data 269
9.2.4 Statistical Treatment of Historical Data 271
9.2.5 Healthy Subpopulation Versus General
Population in Toxicity Sensitivity 271
9.3 CK: Properties and Characteristics 272
9.4 CK IH Toxicology 273
9.4.1 General Toxicology 274
9.4.2 Acute Human Toxicity 274
9.4.3 Acute Mammalian Toxicity 275
9.5 Previous Human Lethality Estimates for CK IH
Toxicity 282
9.5.1 UK: Porton (Unofficial) 282
9.5.2 UK: Health Safety Executive 282
9.6 Data Analysis and Results 283
9.6.1 Data Reduction 283
9.6.2 PS Estimation 284
9.6.3 Linear Regression Analysis 286
9.6.4 Allometric Scaling of Mammalian CK
Lethality 288
9.6.5 TLE and Time–Concentration Relationship 290
9.6.6 Human Lethality Estimates for CK IH 291
9.6.7 Human Severe Effect Estimates for CK IH 291
9.6.8 Comparison of Estimates: Present Chapter
with Previous Work 293
9.6.9 Comparison of CK and AC Human
Lethality Estimates 294
9.6.10 Comparison of CK and AC
Mammalian Lethality 295
9.7 Discussion and Conclusions 298
References 298
Subject Index 307

xvi Contents
Volume 2: Management of Poisoning
Chapter 1 Treatment of Nerve Agent Poisoning 1

Horst Thiermann, Nadine Aurbek, and Franz Worek
1.1 I ntroduction 1
1.2 OP Compounds 2
1.2.1 General Remarks That Are Relevant
for Therapy 2
1.2.2 Toxicology of OP Compounds 3
1.3 Protective Measures and Decontamination 6
1.4 Clinical Picture of Nerve Agent Poisoning 8
1.4.1 Acute Nerve Agent Poisoning 8
1.4.2 Intermediate Syndrome 10
1.4.3 Organophosphate Induced Delayed
Neuropathy 10
1.5 Pretreatment 10
1.6 Differences Between Nerve Agent and OP
Compound Pesticide Poisoning 11
1.7 Therapeutic Regimen of Nerve Agent Poisoning 12
1.7.1 General Considerations 12
1.7.2 Atropine 12
1.7.3 Oximes 16
1.8 Summary and Outlook 30
References 31
Chapter 2 Nerve Agents: Catalytic Scavengers as an Alternative

Approach for Medical Countermeasures 43
Patrick Masson
2.1 I ntroduction 43
2.2 The Scavenger Concept 44
2.3 Endogenous Bioscavengers 46
2.4 Stoichiometric Scavengers 46
2.5 Pseudo-Catalytic Scavengers 48
2.6 Catalytic Scavengers 49
2.7 Requirements for Operational Catalytic Scavengers 49
2.8 Potential Enzymes 52
2.8.1 Phosphotriesterases 52
2.8.2 Engineered ChEs and CaEs 56
2.8.3 Oxidases 59
2.9 Catalytic Antibodies 60
2.10 Artificial Enzymes 60
2.11 Future Directions 60
References 62
Contents xvii
Chapter 3 Nicotinic Receptors as Targets for Nerve Agent Therapy 82
John Tattersall
3.2 Current Therapy for Nerve Agent Poisoning 83
3.3 Potential Benefits of Nicotinic Antagonists in
Nerve Agent Poisoning 84
3.4 Nicotinic ACh Receptors 85
3.5 The Muscle nAChR 86
3.6 Blockers of Neuromuscular Transmission 89
3.7 Effects of AChE Inhibitors at the NMJ 92
3.8 Anti-Nicotinic Effects of Oximes 93
3.9 Optimisation of the Anti-Nicotinic Properties of
Bispyridinium Compounds 94
3.10 Protection Against Delayed Respiratory Failure 97
3.11 Neuronal Nicotinic Receptors 98
3.12 Drugs Acting at Neuronal Nicotinic Receptors 101
3.13 Neuronal Nicotinic Effects in Nerve Agent
Poisoning 102
3.13.1 Mecamylamine 102
3.13.2 Benthiactzine 103
3.14 Summary 103
References 104
Chapter 4 Mustard: Pathophysiology and Therapeutic

Approaches 120
Dirk Steinritz, Frank Balszuweit, Horst Thiermann,
and Kai Kehe

4.2 Chemistry 121
4.3 Toxicokinetics 121
4.4 Clinical Picture 124
4.4.1 Skin 124
4.4.2 Lungs 125
4.4.3 Eyes 125
4.5 Therapeutic Interventions for SM Injury
in Correlation with the Molecular Pathology 126
4.5.1 Search for Novel Therapies, the Animal
Efficacy Rule, Regulatory Requirements
and Their Effect on Research Strategies 128
4.5.2 Alkylation 129
4.5.3 Direct SM Scavengers 131
4.5.4 Inflammation 131
4.5.5 Anti-Inflammatory Therapy 132
xviii Contents
4.5.6 Repair of SM Induced DNA Lesions with a
Focus on PARP 133
4.5.7 PARP Inhibitors 134
4.5.8 SM and Cell Death 135
4.5.9 Limitation of Extrinsic Apoptosis 137
4.5.10 Reactive Species Formation After
SM Exposure 137
4.5.11 Anti-Oxidative Interventions 138
4.5.12 Other Therapeutic Approaches 140
References 146
Chapter 5 Clinical and Laboratory Diagnosis of Chemical

Warfare Agent Exposure 157
Franz Worek, Horst Thiermann, and Timo Wille

5.2 Structure and Mechanism 158
5.3 Clinical Diagnosis 159
5.3.1 Clinical Signs of Nerve Agent Poisoning 160
5.3.2 Clinical Signs After Exposure to Volatile
Nerve Agents 161
5.3.3 Clinical Signs After Exposure to Low
Volatility Nerve Agents 162
5.3.4 Alternative Indicators of Nerve Agent Exposure 164
5.4 Laboratory Diagnosis 164
5.4.1 Cholinesterases as Diagnostic Markers 165
5.4.2 Onsite Determination of Cholinesterase
Activity 166
5.4.3 Therapeutic Monitoring: The Cholinesterase
Status 168
References 172
Chapter 6 Verification of Exposure to Chemical Warfare Agents 179

Robert W. Read

6.2 Biomarkers of Exposure 180
6.2.1 Analytical Methods and Instrumentation 182
6.3 Sulphur Mustard 184
6.3.1 Application to Human Samples 185
6.4 Nerve Agents 194
6.4.1 Application to Human Samples 200
6.5 Other Agents 205
6.5.1 Analogues of Sulphur Mustard 205
Contents xix
6.5.2 Nitrogen Mustards 205
6.5.3 Lewisite 206
6.5.4 Quinuclidinyl Benzilate 207
6.5.5 Phosgene and Hydrogen Cyanide 207
6.6 Further Developments 208
References 208
Chapter 7 The Impact of New Technologies on the Elucidation of

Chemical Warfare Agent Toxicology 219
Jonathan David and Harald John

7.2 Transcriptomics 220
7.2.1 The Use of Microarrays to Study
CWA Toxicology 221
7.2.2 Alternative RNA Based Approaches 231
7.2.3 Transcriptomics Conclusion 233
7.3 Proteomics 234
7.3.1 The Principle Strategy of Proteomics 234
7.3.2 Two-Dimensional Gel Electrophoresis 235
7.3.3 Mass Spectrometry 236
7.3.4 Special Analytical Feature for Relative
or Absolute Protein/Peptide
Quantification 238
7.3.5 Proteomic Research of CWAs 241
7.3.6 Proteomics Conclusion 244
7.4 In silico Approaches 245
7.5 Overall Conclusion 247
References 248
Chapter 8 Chemical Defence Against Fentanyls 259

Christopher D. Lindsay, James R. Riches, Neil Roughley,
and Christopher M. Timperley

8.2 Brief History 260
8.3 Medicinal Uses 263
8.4 Military Interest 265
8.5 Illegal Trade 267
8.6 Moscow Theatre Siege 269
8.7 Resurgence of Military Interest 273
8.8 Biological Effects 273
8.8.1 Inhalation Toxicity 273
8.8.2 Dermal Toxicity 275
8.8.3 Opioid Antagonists 276
8.8.4 Opioid Receptors 279
xx Contents
8.9 Chemistry 280
8.9.1 Properties 280
8.9.2 Pyrolysis 281
8.9.3 Hydrolysis 283
8.9.4 Decontamination 285
8.9.5 Detection 289
8.10 Identification 290
8.10.1 Environmental Samples 290
8.10.2 Biomedical Samples 290
8.11 Conclusions 293
References 294
Subject Index 314

Chapter 1
Development, Historical Use

and Properties of Chemical
Warfare Agents
Robin Black*
*E-mail: rjcpblack@gmail.com
1.1 Introduction
The year 2014 was the centenary of the commencement of the 1914–18 war
[World War I (WWI)], a conflict that resulted in more than 20 million deaths
and unprecedented numbers of casualties. A notorious development of that
conflict was the widespread use of chemical weapons, manufactured on an
industrial scale. Since 1914 more than 700 000 tonnes of chemical agents
have been produced by various nations but, fortunately, since 1918 the use of
chemical weapons in warfare has been the exception rather than the rule. Nev-
ertheless, their use in conflicts in Iraq in the 1980s and more recently in Syria
in 2013, particularly against unprotected civilian populations, has served as
a reminder that the dangers still exist, even though a near comprehensive
treaty, the Chemical Weapons Convention (CWC), entered into force in April
1997.1 This chapter provides the reader with a brief history of the develop-
ment and use of chemical weapons, a summary of the physicochemical prop-
erties that determine the primary hazard posed by chemical agents and how

1
2 Chapter 1
they might be used, and finally an overview of the main classes of chemical
warfare (CW) agents known to have been weaponised, or which are known or
suspected to have progressed to advanced development at some stage.
1.2 Brief History of CW

1.2.1 Prior to 1914
Dating from ancient times there had been sporadic exploitation of toxic
chemicals for use in warfare; examples include poisoned arrows, the burning
of sulfur to produce asphyxiating fumes, and the use of crude irritants to
drive defenders into the open. The lachrymator ethyl bromoacetate was used
in France for law enforcement operations from 1912–1914. More detailed
historical accounts are available.2–4 These relatively minor and small scale
uses of chemicals had been sufficient to warrant the drafting of prohibitive
articles as part of two conventions on the conduct of land warfare. The 1899
Hague Convention forbade the use of poison or poisoned weapons, and
included a declaration that parties would ‘abstain from using projectiles the
sole object of which is the diffusion of asphyxiating or deleterious gases’;
these were reaffirmed in the 1907 Hague Convention.5
The most significant development in the decades before WWI was the
rapid expansion of the chemical industries of the main protagonists. Com-
modity and other chemicals were being produced in large industrial plants
on multi-tonne scales, and this laid the foundation for the subsequent pro-
duction of CW agents on a scale suited to WWI battlefields.
1.2.2 The 1914–18 War (WWI)

1.2.2.1 Overview
The advent of the tactical and strategic large scale use of chemical weapons
occurred almost one year into WWI. Major land battles, mostly in Europe
and Russia, involved thousands of soldiers entrenched in high concentra-
tions on vast, muddy and relatively flat areas of land. These attritional battles
often continued for weeks or even months, many resulting in stalemate. The
high concentration of relatively static entrenched combatants, initially with
little protection, was seen as an ideal target for chemical weapons, offering a
possible means of breaking the stalemate.
The main CW agents used in WWI were various irritants, chlorine, phos-
gene, and in the later stages sulfur mustard.6–9 In addition to their direct
effects, the psychological effects of fear of exposure proved to be a significant
factor. It is estimated that approximately 125 000 tons of agent were used,
resulting in around 1.25 million casualties, of which 90 000–100 000 were
mortalities. Chemical agents accounted for approximately 4% of combat
deaths in WWI.6 The effectiveness of chemical attacks was gradually reduced
as protective countermeasures were introduced.
Development, Historical Use and Properties of Chemical Warfare Agents 3
1.2.2.2 Irritants
The first minor and ineffective use of chemical weapons in WWI occurred in
August 1914 when French forces fired 26 mm grenades containing the lachry-
mator ethyl bromoacetate at German troops. A much larger scale dissemination
of an irritant occurred in January 1915, when German forces fired 18 000 artil-
lery shells containing xylyl bromide at Russian positions west of Warsaw during
the battle of Bolinov. This too was largely ineffective because the agent froze
in the cold conditions of the eastern front. Use of various lachrymatory and
respiratory irritants continued, with more than twenty being used in WWI.10,11
1.2.2.3 Chlorine
The seminal incident generally regarded as the advent of large scale tactical
CW occurred at the battle of Ypres, Belgium, on 22 April 1915. German troops
released 168 tons of the industrial gas and lung injurant chlorine from 5730
cylinders, late in the afternoon and in a slight breeze towards entrenched
allied soldiers. Chlorine is much denser than air, and lingered close to the
ground and trenches; casualty numbers are uncertain but probably totalled
several thousand.8 Ironically this first use was more effective than German
forces had anticipated, and they failed to capitalise on the resultant breach
in the allied defences. Further releases of chlorine occurred on both the west-
ern and eastern fronts in 1915. It was used much less successfully by British
forces at Loos in October 2015, when a late change of wind direction moved
the toxic cloud back towards entrenched British soldiers.8 As well as relying
on wind direction for dissemination when released from cylinders, chlorine
was readily detected by its familiar odour and the visible yellow–green cloud.
Basic protective countermeasures were developed and chlorine on its own
was gradually abandoned in favour of phosgene, although use continued in
admixture with phosgene and chloropicrin.12
1.2.2.4 Phosgene
The year 1915 saw the first use, initially by France, of the highly volatile indus-
trial chemical phosgene.12 Phosgene is three to four times more potent than
chlorine as a lung injurant and was to become the most effective of the lethal
agents used in WWI, causing approximately 85% of the deaths from expo-
sure to chemical agents. Phosgene was more insidious than chlorine, being
colourless, its odour (fresh mown hay) less obvious, and onset of serious overt
effects (pulmonary oedema) delayed for several hours. It was initially released
from cylinders alone or mixed with chlorine, but was later liquefied and used
in projectiles. Approximately 36 000 tons was manufactured for use in WWI.
Germany preferred to use diphosgene (trichloromethyl chloroformate), which
has a similar toxicity to phosgene but is a liquid [boiling point (bp) 128 °C]. It
was therefore more persistent, and more easily handled and loaded into pro-
jectiles.8 Several less effective lung injurants were used in WWI.12
4 Chapter 1
1.2.2.5 Sulfur Mustard
The most effective chemical agent of WWI proved to be the liquid vesicant
sulfur mustard, which caused more casualties than all of the other agents
combined, even though it was introduced late in the war.6,8 Its first use was
by Germany against French forces at Ypres on 12 July 1917, producing an
estimated 15 000 casualties. Development and use by British, French and
US forces soon followed. Not only did sulfur mustard cause serious blister-
ing by contact with the skin, its vapour caused serious damage to the eyes
and lining of the respiratory tract. One of the enduring images of WWI is a
chain of walking mustard casualties with bandages over their damaged eyes.
Although only around 2% of mustard casualties were mortalities (mostly
from secondary lung infections), the large number of injured soldiers caused
huge logistic problems for medical treatment.
1.2.2.6 Other Agents
Both sides experimented with other agents, mostly volatile noxious industrial
chemicals but also some solid agents.8,13,14 Examples of eye irritants were bro-
moacetone, bromobenzyl cyanide, chloropicrin and ethyl iodoacetate. Chlo-
ropicrin, which was first used by Russian forces, caused a significant number
of deaths when used at high concentration. More toxic chemicals included
hydrogen cyanide (HCN), cyanogen chloride and hydrogen sulfide.15 HCN was
largely ineffective as a lethal agent. It is slightly less dense than air and rapidly
dispersed, making it difficult to sustain effective dosage levels with the muni-
tions available. The French later used the denser cyanogen chloride. Several
respiratory irritant arsenicals, some also with vesicant action, were used in the
later stages of WWI, e.g. ethyl-, methyl- and phenyldichloroarsine, diphenyl-
chloroarsine (DA) and diphenylcyanoarsine (DC).11 DA and DC irritated eyes
and mucous membranes of the nose and throat, causing sneezing and cough-
ing, and induced vomiting. As solids disseminated as crude particulate aerosols
rather than vapour, they were conceived by Germany as possible mask breakers,
and used in combination with more toxic lung injurants. They were countered
by inclusion of a mechanical filter in the mask. Two additional arsenicals, the
newly developed vesicant lewisite, and the respiratory irritant/vomiting agent
adamsite (DM), would have become available had the war lasted beyond 1918.
1.2.3 The Inter-War Years

Following the experiences of WWI, a strong desire to prohibit the use of poison
gas led to the 1925 Geneva Protocol, which prohibited ‘the use of asphyxiating,
poisonous and other gases, and of all analogous liquids, materials and devices’
(and bacteriological methods of warfare).16 It did not prohibit production of CW
agents and several signatories reserved the right to retaliate in kind following
first use against them. Notwithstanding this protocol, further development of
existing and new chemical agents, plus more effective munitions and delivery
systems, continued.17 Examples of agents included the further development of
18
the arsenicals lewisite and DM, the vesicant nitrogen mustards, and the ‘net-
tle gas’ or urticant phosgene oxime.19 The latter causes severe pain and lesions
of the skin and was conceived as a possible means of forcing removal of res-
pirators; fortunately it has poor stability. The lachrymator 1-chloroacetophe-
none (CN) was developed as the standard riot control and harassing agent by
the USA and adopted in other countries.20 DM was also used as a riot control
agent (RCA) during this period in the USA and other countries, and became
a significant component of military arsenals. By the onset of World War II
(WWII) a broader variety of munitions for CW agents was available as well as
the means to deliver them from artillery and aircraft. Research continued into
more effective defensive countermeasures (detection, protective clothing, res-
pirators, medical countermeasures and decontaminants).
CW agents were reportedly used in several localised, mainly colonial, con-
flicts in the interwar years.21 Of particular note was the first major use of
airborne delivery when Italy sprayed sulfur mustard from aircraft during its
invasion of Abyssinia in 1935/36. A year later Japan began using chemical
weapons on the Chinese mainland.
The most important development during this period, which occurred
during the build up to WWII, was the chance discovery and military develop-
ment of organophosphorus nerve agents in Germany. In 1936, the chemist
Gerhardt Schrader, working in the laboratories of the industrial conglomer-
ate IG Farben, synthesised an experimental organophosphorus insecticide
that proved to be an order of magnitude more toxic to animals than any agent
used in WWI. The discovery was disclosed to the military, and the chemical
was developed in secrecy into the CW agent named tabun.22 This develop-
ment was to dominate offensive and defensive chemical weapon research
and development for the next 40–50 years.23
1.2.4 The 1939–1945 War (WWII)

In spite of fears to the contrary, chemical weapons were not used in Europe in
WWII. One theory was that Hitler had an aversion to CW from his experiences
in WWI. Perhaps a more convincing argument was that first use would simply
result in retaliation in kind. The major protagonists had built large stockpiles
of chemical weapons in the build up to and during WWII, chiefly sulfur mus-
tard, small quantities of nitrogen mustards, lewisite, various other arsenicals,
phosgene, HCN and cyanogen chloride.8 The UK had plans to use the per-
sistent agent sulfur mustard on the beaches in the event of a German land inva-
sion. HCN in the form of Zyklon B, a commercial pesticide formulation of HCN
adsorbed onto a solid support, was used to kill up to a million people in the gas
chambers by Nazi Germany. Japan continued to use chemical agents on main-
land China, chiefly sulfur mustard, and possibly lewisite, DC and phosgene.
Germany produced approximately 12 000 tons of tabun for weaponisation
before and during WWII. Systematic molecular modification of tabun led to
the more potent and volatile nerve agent sarin, which was later to become one
of the major components of modern chemical arsenals.8,23 A small quantity of
sarin (estimated variously from 500 kg to 10 000 tons) was produced on a pilot
6 Chapter 1
plant scale in Germany. A less volatile analogue of sarin, given the name soman,
was developed by Germany towards the end of the war (see Section 1.4.4).
Britain and the USA had also researched organophosphorus compounds
as potential CW agents during this period but the leading candidate, O,O-di-
isopropyl fluorophosphate (DFP), was significantly less potent than tabun.24
1.2.5 Post WWII and the Cold War Years

Following the end of WWII, details of the German programme were obtained
by allied intelligence. The tabun plant at Dyhernfurth was dismantled and
rebuilt in Russia, and small stocks of tabun (given the US military designator
GA, G = German) were acquired by the USA and UK. Interest in tabun then
gradually waned as sarin (GB) became the favoured nerve agent. The imme-
diate post war years saw the intensive exploration of analogues of sarin and
soman (GD), by the UK, USA, Russia, France and other nations.25 Sarin was
later weaponised and stockpiled by the USA and the Soviet Union; soman was
weaponised and stockpiled by the Soviet Union neat and in a thickened form
as a more persistent nerve agent. Other nations, e.g. France, built pilot plants
and acquired small stockpiles of sarin.
History was repeated in 1952 when a low volatility organophosphorus pes-
ticide, later named amiton, was discovered in the plant protection laborato-
ries of Imperial Chemical Industries (ICI) in the UK.26 The notable feature
of amiton was its unusually high percutaneous toxicity in rodents and rab-
bits. The discovery was disclosed to the UK Porton Down laboratory, where
molecular modification led to the development of the low volatility V (ven-
omous) agents in a collaboration with the USA and Canada. Similar agents
were developed independently in the Soviet Union. The USA weaponised the
analogue designated VX, based mainly on a combination of its toxicity and
storage stability. The Soviet Union weaponised an isomeric analogue code-
named R-33, usually now referred to as RVX or VR. These agents were devel-
oped and stockpiled with the possibility of a major conflict between western
and Soviet forces, e.g. on the northern plains of Europe, sarin as a rapid act-
ing non-persistent casualty producing agent, and V agents and thickened
soman as persistent casualty producing and disruptive agents, for terrain
denial, and for attacking rear logistical sites.27 Large stockpiles of sulfur mus-
tard were retained. The relative importance of these agents is reflected in the
later declarations to the CWC (see Table 1.1).
From the 1970s, the USA and Soviet Union moved away from producing
unitary agents (i.e. the final product) for weaponisation because of the haz-
ards of production, storage and transport, plus the high cost of destruction
when required. Both started to develop binary weapons, wherein two reactive
precursors are placed in a munition separated in rupturable compartments.
On firing, the barrier is broken and the rotation caused by rifling of the muni-
tion results in rapid mixing of the precursors to produce the agent during the
time of flight.28 The USA developed binary munitions for sarin and VX; the
Soviet Union developed binaries for sarin and RVX.29,30 Binaries may also be
advantageous if the agent has poor storage stability.
Table 1.1 Quantities
of chemical agents declared to the OPCW as of 31 December
200145.
Agent Total declared (tonnes)a
Sulfur mustard 13 839
Sulfur mustard/T mixture 3536
Sulfur mustard/lewisite mixture 345
Lewisite 6745
Sarin 15 048
Soman 9175
VX 4032
RVX 15 558
Tabun 2
a
Rounded to the nearest tonne; excludes declarations ≤1 tonne.
Major advances were made in defensive countermeasures during this period,

such as manual and automated detectors (for warning and monitoring con-
tamination), protective clothing and respirators, decontaminants and medical
countermeasures, the latter particularly against nerve agents.31 It was therefore
not surprising that continuing research into potential new agents, both offen-
sive research and defensive threat agent assessment, began to focus on possible
means of defeating these countermeasures. Examples were canister penetrat-
ing volatile organofluorine agents (e.g. perfluoroisobutene, trifluoronitroso-
methane),29,30 fast acting vomiting agents to force removal of the respirator,27
and intermediate volatility nerve agents that could penetrate semi-permeable
suits.29,30 Most of these experimental agents had significant shortcomings.
CN was gradually replaced by the more effective and less toxic irritant
2-chlorobenzilidene malononitrile (CS) for law enforcement, riot control and
military use; pepper spray was widely introduced for self defence or as an aid
to arrest. The use of irritants for civilian riot control increased substantially
during these years in an age of widespread protest.
From the 1950s there were programmes in several countries aimed at
exploiting the rapidly increasing pace of pharmaceutical and veterinary drug
discovery, particularly centrally acting drugs, for military or law enforcement
purposes.20,32 In 1963 the USA weaponised the anticholinergic compound
3-quinuclidinyl benzilate (BZ) as a military incapacitating agent.
1.2.6 The Middle East

Chemical weapons have been used in at least four conflicts in the Middle
East, and it remains the most volatile region with regard to CW. Sulfur mus-
tard and phosgene were reported to have been used by Egypt in its interven-
tion in the North Yemen civil war in the mid-1960s.21 Alleged use of sarin in
the Yemen was not confirmed. Iraq used sulfur mustard, tabun and sarin in
the conflict with Iran from 1980–1988, causing an estimated 150 000 casual-
ties.33–35 Iraq also used sulfur mustard and sarin against its indigenous Kurd-
ish population, the most infamous occurrence being in the city of Halabja
in March 1988, where between 3000 and 5000 deaths were reported, mostly
8 Chapter 1
36
unprotected civilians. Multiple agents appear to have been used in Halabja
including sulfur mustard and nerve agents—probably sarin but no analyt-
ical evidence confirming the lethal agent used has been published. Four
years later, retrospective investigations of an attack on the Kurdish village
of Birjinni provided the first analytical evidence corroborating the use of
sarin and further use of sulfur mustard.37,38 In the aftermath of the Gulf War
with Iraq, there were incidents of crude use of chlorine by insurgents. Most
recently (2013) the use of sarin was confirmed in the internal conflict in Syria,
although the UN investigative mission did not identify the perpetrators.39
1.2.7 Terrorism
Although there have been incidents of small scale terrorist or criminal use
of irritants, poisons and powders contaminated with the toxin ricin, the per-
ceived threat of moderate scale dissemination of chemical agents by terror-
ists has not materialised other than in Japan in the 1990s.40 In Matsumoto
City, June 1994, members of the Aum Shinrikyo religious cult disseminated
vaporised sarin from a van towards an apartment block, targeting three
judges overseeing a land dispute with the cult. The judges survived but seven
others were killed with around 270 casualties. The second use of sarin by
Aum Shinrikyo was to have a major impact on home security programmes
throughout the world.40 On 20 March 1995, an estimated 20 kg of crude sarin
was released by puncturing plastic bags containing the agent on trains on
five subway lines converging towards government offices and the central
police headquarters. The attack resulted in 12 deaths with approximately
1100 serious casualties. Up to 4000 mildly exposed personnel or ‘worried
well’ presented themselves to hospitals, and the attack left a psychological
imprint on many thousands of people in Japan and elsewhere. The main
reasons for the low number of deaths were the crudeness of the agent and
rather slow method of dissemination. The cult also used VX in an assassina-
tion.41 In some aspects Aum Shinrikyo was an exception in that it was a large
and wealthy organisation operating for many years with a degree of impunity
within a developed nation, a situation that is much less likely today.
With the increasing terrorist threat of hostage taking and aircraft hijack-
ing, the search for potent knockdown incapacitating chemicals for count-
er-terrorist operations continued in several countries. In 2002, Russian
Special Forces ended a siege of a Moscow theatre by disseminating an aero-
sol containing two analogues of the opioid analgesic/anaesthetic fentanyl
into the theatre.42,43 At least 130 out of the 800 hostages plus 40 terrorists
died in the operation, most from exposure to the agent.
1.2.8 Chemical Weapons Convention

As described above, massive stockpiles of chemical weapons (approximately
70 000 tonnes declared by 2001), mainly vesicants and nerve agents, were
accumulated in the cold war years, mostly by the USA and the Soviet Union.
After years of negotiation, a near comprehensive chemical weapons treaty,
the CWC, was opened for signature in April 1993 and entered into force in
October 1997.1,44 Unlike previous treaties, which attempted unsuccessfully to
prevent first use, the CWC prohibits the development, manufacture, stockpil-
ing and use of toxic chemicals in warfare. Furthermore, it requires all stock-
piles to be declared and destroyed. The deadline for the latter was initially 2007,
but this was extended first to 2012 and now to 2020 because of the technical
and economic complexities of destruction. A total of 190 of the UN recognised
nations have ratified the CWC, only six exceptions remain. As of December
2014, Angola, Egypt, North Korea and South Sudan had not signed the Con-
vention, and Israel and Myanmar had signed but not ratified. Following recent
conflicts, CW agent possessor states Iraq, Libya and Syria have signed and rat-
ified the Convention. Table 1.1 summarises the total declarations of agents to
the Organisation for the Prohibition of Chemical Weapons (OPCW) by 2001,
underlining the importance of sulfur mustard and nerve agents.45
1.3 C
lassification, Properties and Modes of Use of
CW Agents
1.3.1 Classification
CW agents have been classified in several ways.46,47 They can be grouped sim-
ply by their predominant gross effect at realistic concentrations:

- Lethal, tissue damaging (casualty producing), irritant (harassing),
incapacitating.

Alternatively, they may be classified more specifically according to their
main physiological effects:

- Vesicants (blister agents), lung injurants (choking agents), blood
agents, nerve agents, irritants (skin, eye and respiratory), incapaci-
tants.

A third way of classifying agents is according to their physicochemical
properties:

- Non-persistent (moderate to high volatility), persistent (low volatility).
1.3.2 Physicochemical Properties

1.3.2.1 Gaseous and Liquid Agents
Physicochemical properties are a major determinant of the hazard (as
opposed to the toxicity) of an agent, and hence the most likely mode of
use.46–48 Although the terms mustard gas, nerve gas, etc. are in common
usage, most CW agents are liquids. Efficient dissemination is key to the effec-
tiveness of a chemical weapon. Irrespective of the agent’s properties, it needs
to be dispersed evenly over the target area.28
10 Chapter 1
Non-persistent agents (e.g. sarin, phosgene and HCN) have moderate to
high vapour pressure and readily vaporise; the least persistent are gases at
ambient temperatures. The major portal of entry is the lung, although the
eyes may also be important. Non-persistent agents have been disseminated
using modified conventional munitions as a mixture of vapour, aerosol and
small droplets, according to their vapour pressure and the energy used for
dissemination. They soon disperse on the battlefield and need to be dissem-
inated in proximity to the ground. Early evening to early morning is gener-
ally the most effective time to disseminate non-persistent agents, when air
temperature stratification is neutral or inverted (coldest nearest the ground),
and turbulence is minimal. Non-persistent agents would rarely (e.g. under
very cold conditions) need to be decontaminated in the field; some are rap-
idly degraded in the environment. The general military philosophy has been
that a non-persistent agent would be the most effective for an on- or near-tar-
get attack to cause rapid casualties, particularly in a surprise attack before
defensive countermeasures are in place, and where the attacker may seek to
occupy the area attacked. Sarin is the most important non-persistent agent;
onset of effects by inhalation occurs in minutes. Such an agent is also attrac-
tive to terrorists because it is easier to disseminate compared with persistent
agents. The most volatile non-persistent agents, e.g. phosgene, chlorine
and HCN, are generally regarded as obsolete because it would be difficult to
achieve effective concentrations over a modern dispersed battlefield. They
could be used effectively against unprotected civilians.
Persistent agents have low vapour pressures and most produce insufficient
vapour concentration to cause large numbers of casualties through inhalation.
Their major portal of entry is through the skin. With the most important per-
sistent agents, i.e. sulfur mustard and V agents, onset of effects is slow (up to
several hours) by this route of exposure. Unless aerosolised, a persistent agent
is most likely to be used in the form of droplets for contaminating the ground
and equipment, rear logistical sites and supply routes. Against well protected
and trained personnel they are predominantly disruptive, forcing defenders
into individual protective equipment, with the attendant physical, physiologi-
cal and psychological impositions, along with impaired communication. The
most effective persistent agents have some resistance to environmental degra-
dation. They may persist on the battlefield for days or even weeks, depending
on weather conditions (temperature, wind and precipitation).28 Wet and windy
conditions can reduce persistence from days to hours. Decontamination is
targeted mainly at persistent agents. Persistent agents can be disseminated
from moderately high altitude, particularly when thickened by the addition
of a few percent of a polymer to prevent dispersion before the droplets reach
ground level. Thickening also improves the ballistics of munitions and makes
decontamination more difficult. The Soviet Union declared thickened sulfur
mustard, lewisite, soman and V agent during a confidence building visit to the
Shikhany proving ground during negotiations towards the CWC.29
There is of course no clear demarcation between persistence and non-per-
sistence, with a continuum of volatilities across a broad spectrum, and with a
strong dependence on the temperature of the environment. Chemicals with
intermediate volatility can be very effective, the prime example being sul-
fur mustard. Depending on weather conditions, sulfur mustard can produce
sufficient vapour to cause casualties through inhalation, eye exposure and
contact with moist sensitive areas of the skin, but can present a persistent
contact hazard on the ground when present as droplets (see Section 1.4.3.1).
Unless thickened, soman is rather too volatile to be an effective intermediate
volatility agent (IVA), but the less volatile cyclosarin (GF) and 2-methyl GF fall
within the intermediate volatility range. Boiling points and volatilities of the
main agents at 25 °C are shown in Table 1.2.49 Volatility is defined as the max-
imum concentration of vapour in equilibrium with liquid agent in a confined
space and at a defined temperature; it is derived from vapour pressure data.
In an open space such as a battlefield only a small percentage of this value is
likely to be achieved. Volatility is highly dependent on temperature, with a 10 °C
increase above 20 °C approximately doubling the volatility.
Other physical properties are also important. Surface tension determines
the extent of spreading (e.g. oil versus water). Most liquid CW agents resem-
ble organic liquids or oils, and have a significantly lower surface tension than
water. They tend to spread on surfaces, getting into parts that are difficult to
decontaminate with water-based decontaminants. Viscosity determines thick-
ness, how readily an agent sticks to surfaces, and drop size on dissemination.
1.3.2.2 Solid Agents
Solid agents are a special case in that they are generally dispersed as aerosols,
fine particles with particle sizes in the respiratory range (1–10 µm diameter).48
Particulate aerosols are most efficiently generated thermally or pneumatically,
targeting the lung as the primary portal of entry. One of the most efficient
and rapid means of disseminating solid agents is using multiple pyrotechnic
sub-munitions. The agent is mixed with a pyrotechnic formulation, it is rap-
idly (within seconds) vaporised at high temperature, and immediately con-
densed to a particulate aerosol in the cold air. In the 1960s the USA designed
cluster bombs intended to be filled with multiple pyrotechnic sub-munitions
Table 1.2 Boiling

points and volatilities of CW agents49.
Agent bpa (°C) Volatility at 25 °C (mg m−3)
Phosgene 7.8 7.46 × 106
HCN 25.5 1.10 × 106
Sarin 150 18 700
Lewisite 196 3860
Soman 198 3930
Sulfur mustard, HD 218 906
Cyclosarin, GF 228, 239b 898
Tabun 248 497
HN-3 257 120
VX 292 12.6
a
Most of the higher boiling agents decompose, bp values are extrapolated.
b
Quoted by Marrs et al.31
12 Chapter 1
50
containing the irritant CS or the incapacitant BZ. Such cluster munitions
were designed to disseminate effective concentrations over an area up to
around 1 hectare within masking time. Persistence of aerosolised solid agents
is generally considered to be low, although the residual hazard from impacted
or deposited aggregated solid agent has not been clearly defined.
1.3.3 Ease of Production

A chemical property that has been one of the major factors in determining
the extent of proliferation of CW agents is the ease of production.51–55 To a
crude rule of thumb, around 1 tonne of agent is considered the minimum
required for a small but militarily significant attack, although disruption can
be caused by smaller quantities, or even by a credible threat of use. For ter-
rorist purposes, considerably lower quantities could be effective, particularly
if panic and publicity are the desired effects.
In WWI, chlorine, phosgene and some other industrial chemicals were avail-
able in multi-tonne quantities. Sulfur mustard was easily synthesised from
industrially available precursors by a one or two stage process.55 Nerve agents
present a somewhat greater challenge but should not be too difficult for a nation
with a moderately developed chemical industry. Nerve agents require between
three and eight stages depending on the agent and precursors available, the ease
of synthesis being tabun > sarin > cyclosarin > soman > RVX/VX.51–53 It is instruc-
tive to follow the order of development of chemical agents by Iraq in the 1980s.
Sulfur mustard was the first agent to be produced and weaponised, followed in
order by tabun, sarin, sarin-GF mixture, and finally VX, which proved problem-
atic. This is in the order of complexity of production. Cyclosarin was selected
rather than soman because of the greater availability of a key precursor, cyclo-
hexanol, rather than pinacolyl alcohol required for the production of soman.
Stability on storage is an important aspect of chemical production.54 US
and Soviet Union cold war stockpiles were produced for an uncertain future
conflict. Long term storage stability (e.g. 10–20 years) was therefore imper-
ative. In contrast, Iraq in its conflict with Iran tended to produce and use.
Stability is partly dependant on the purity of the agent and on the addition
of stabilisers, particularly for nerve agents. Decomposition of stored agents
tends to be self-accelerating. Reaction of nerve agents and sulfur mustard
with traces of moisture releases hydrofluoric or hydrochloric acid, which
accelerate further degradation. Stabilisers such as diisopropylcarbodiimide
or tributylamine are added to scavenge any moisture or acid formed.
1.4 Main Classes of Chemical Agents

1.4.1 Lung Injurants (Choking Agents)
1.4.1.1 Chlorine
Chlorine, Cl2, is a corrosive industrial gas (bp −34 °C) with a pungent and
characteristic odour, and yellow–green colour.12 It is produced industrially by
the electrolysis of brine and used extensively in the production of industrial
chemicals and consumer products, and for disinfection. Chlorine is a strong
oxidizing agent and is destructive towards lung and eye tissues, but a rela-
tively high concentration is required to cause death. Although obsolete as a
military agent after WWI, there have been isolated incidents of crude oppor-
tunistic chlorine dissemination by insurgents in Iraq and Syria, and allega-
tions of chlorine use in Bosnia in 1993.
1.4.1.2 Phosgene and Diphosgene

Phosgene, COCl2 (military designator CG), is a colourless gas (bp 8 °C) except
at low ambient temperatures, with an odour of freshly mown hay.12 It is pro-
duced on a multi-million tonne scale from carbon monoxide and chlorine,
and is used for the manufacture of a broad range of chemical products. As
a lethal CW agent it is approximately 3–4 times more potent than chlorine
and, as described in Section 1.2.2.4, was the most effective lethal agent used
in WWI. Its effects are much more insidious than chlorine, initially causing
mild irritation of the throat followed by a latent period of up to 24 hours to
induce pulmonary oedema, by which time its effects are life threatening. It is
assumed that phosgene reacts with various nucleophilic sites on macromol-
ecules in the lung but the precise mechanism of action is unknown. Effec-
tive concentrations of phosgene would be difficult to sustain on a modern
dispersed battlefield. As a military agent it is generally regarded as obsolete
although it could still be used effectively against unprotected civilians.
Diphosgene or trichloromethyl chloroformate, ClCO2CCl3, (DP) is a volatile
liquid (bp 128 °C).12 Industrially it is used in the same way as phosgene. It has
a similar toxicity to phosgene but is more persistent, and offers the advan-
tage of being more easily handled. The name diphosgene derives from its
disproportionation into two molecules of phosgene on heating or catalysis.
1.4.1.3 Perfluoroisobutene
Perfluoroisobutene, (CF3)2C=CF2 (bp 7 °C), included in Schedule 2 of the
CWC, is also a lung injurant that causes pulmonary oedema. It is a by-prod-
uct of Teflon production. Like phosgene it is a reactive electrophile. It is not
known to have been weaponised but was studied as a potential hydrophobic
canister penetrant.56
1.4.2 Blood Agents

1.4.2.1 Hydrogen Cyanide
HCN (AC) is a colourless liquid or gas (bp 25.7 °C).15 Its odour of bitter
almonds is not perceptible by some people, although a bitter taste in the
mouth may be evident. It is produced industrially by a catalytic reaction of
methane with ammonia. Its many uses include chemical and polymer pro-
duction, in electroplating, and as a fumigant pesticide and rodenticide. Liq-
uid HCN is prone to violent polymerisation and requires stabilisation when
14 Chapter 1
stored in bulk. Unlike chlorine and phosgene, HCN vapour is marginally less
dense than air and its sparse use in WWI was largely unsuccessful because it
was difficult to obtain effective concentrations with the munitions available.
In stark contrast to phosgene, its effects at lethal concentrations are rapid
in onset (seconds to minutes). It prevents cells, including blood cells, from
utilising oxygen by inhibiting the enzyme cytochrome C oxidase.57
1.4.2.2 Cyanogen Chloride
Cyanogen chloride, ClCN (CK), is also a volatile liquid or gas (bp 13.1 °C);
it was used more successfully than HCN in WWI mainly due to its higher
density.15 It is less potent than HCN as a lethal agent, but is a respiratory irri-
tant at sub-lethal exposure concentrations and is thus more easily perceived.
Cyanogen chloride is widely used in the chemical industry. HCN and cyano-
gen chloride are regarded as obsolete CW agents.
1.4.3 Vesicants (Blister Agents)

1.4.3.1 Sulfur Mustard
Sulfur mustard, bis(2-chloroethyl)sulfide (Scheme 1.1), military designator
in distilled form HD, is a medium to low volatility liquid (bp 213 °C) with a
consistency resembling a light oil, and an odour (due to impurities) reminis-
cent of mustard or garlic. It was first synthesised, and its vesicant properties
noted, in the 19th century, but was not fully characterised until its devel-
opment as a CW agent by German scientists during WWI. After devastating
use in WWI, sulfur mustard has remained one of the major threat agents, as
illustrated by the quantities shown in Table 1.1 declared to the OPCW (and
these figures exclude late signatories to the CWC, Iraq, Syria and Libya).
Sulfur mustard is very easily made from single stage processes from
industrial chemicals (sulfur monochloride or dichloride plus ethylene; or
thiodiglycol plus hydrogen chloride).55 It has close to ideal physicochemical
properties for a disruptive CW agent except for a high freezing point (melting
point 14 °C when pure). It is generally persistent when dispersed as droplets
posing a prolonged contact hazard but, under moderate temperatures, still
produces sufficient vapour to damage eyes, lungs and sensitive areas of the
skin. Larger sized drops can be much more persistent than predicted from the
vapour pressure. This results from oligomers being formed at the air–liquid
interface. Mustard reacts with water at the two electrophilic carbon atoms,
Scheme 1.1 Structures

of sulfur mustard and homologues T and Q.
58
and with oxygen at the nucleophilic sulfur atom. Large ‘footballs’ of sulfur
mustard, protected by a polymeric outer coating, are still being dredged up
by fisherman from stockpiles dumped in the Baltic Sea after WWII.59
Although sulfur mustard reacts rapidly with water when in solution, its
degradation in the environment is limited by its very low affinity for water
(solubility 0.092 g/100 g at 22 °C).19 The latter property also makes it one of
the more difficult agents to decontaminate, and more robust for long term
storage compared with nerve agents.
The freezing point of sulfur mustard has been reduced in a number of ways
to prevent the agent from solidifying in weapons in cold weather. In WWI,
mustard was mixed with various solvents, e.g. carbon tetrachloride and ben-
zene. In WWII, Britain produced it from thiodiglycol and hydrogen chloride
as a 6 : 4 mixture with the oligomer ‘T’ (Scheme 1.1), also known as O mus-
tard. T has somewhat greater vesicant activity than sulfur mustard, is less vol-
atile and more persistent. Other nations mixed mustard with lewisite, which
also accelerated the onset of effects and increased the vapour hazard.
Medical treatment of mustard injuries causes major logistical problems,
and military casualties may be unable to perform duties for weeks or per-
manently. It seems remarkable that there is still no effective treatment for
mustard lesions other than symptomatic and palliative treatment. One of
the reasons is that, unlike nerve agents, which undergo a selective and cat-
alytic chemical reaction with the enzyme acetylcholinesterase (analogous to
Erlich’s ‘magic bullet’ in a drug context), sulfur mustard is more akin to a
‘shotgun’, alkylating many nucleophilic groups on DNA, proteins and other
macromolecules. Thus far, the key targets mediating lesions (other than DNA
for carcinogenicity) have not been identified.
A number of analogues and oligomers of sulfur mustard with similar or
slightly greater vesicant activity were developed as agents of lesser impor-
tance (Scheme 1.1). The homologue Q, sesquimustard, is a solid (melting
point 57 °C) and like T was mixed with sulfur mustard.
1.4.3.2 Nitrogen Mustards
The three nitrogen mustards HN-1, HN-2 and HN-3 (Scheme 1.2) are tertiary
amines substituted with 2-chloroethyl groups similar to sulfur mustard. As free
bases they are low volatility liquids, generally with poor stability, but form more
stable water soluble solid hydrochloride salts.18 They were partially developed
as CW agents during the 1930s but there has been no confirmed use. In WWII
Germany produced 2000 tons of HN-3; the USA produced approximately 100
tons of HN-1 in a pilot plant.3 The most important of the N-mustards is HN-3,

of nitrogen mustards.
16 Chapter 1
which is very easily made from the widely used industrial chemical triethanol-
amine by chlorination with thionyl chloride. It is more stable on storage than
HN-1 and HN-2, and its vesicant activity as a liquid approaches that of sulfur
mustard. HN-3 (bp 257 °C) is significantly less volatile than sulfur mustard and
the vapour hazard is low except under very hot conditions. It might therefore
be more effective than sulfur mustard as a persistent agent in hot climates.
HN-2 has been used to treat some types of cancer.
1.4.3.3 Lewisite
Lewisite, CHCl=CHAsCl2, 2-chlorovinyldichloroarsine, named after its dis-
coverer W. L. Lewis, was produced by the USA and shipped to Europe in 1918,
too late to be used in WWI. Between the wars it was also produced by Japan
and the Soviet Union. It is relatively easily made from arsenic trichloride and
acetylene, although the process is technically more difficult than the pro-
duction of sulfur mustard. Lewisite is more volatile (bp 190 °C) than sulfur
mustard and hence it is less persistent; it also appears to be more sensitive to
environmental moisture. In contrast to sulfur mustard, its initial effect (skin
pain or irritation) is almost instant, and blisters appear within a few hours.19
There has been no confirmed instance of use, although Japan is suspected of
having used lewisite in China in WWII. In addition to being stockpiled as a
neat agent, lewisite was mixed with sulfur mustard to speed up the onset of
action and to depress the freezing point of the latter.
Several other liquid arsenicals with vesicant, lung and eye damaging effects
were developed and used in WWI. Examples are methyl-, ethyl- and phe-
nyl-dichloroarsine (MD, ED and PD) known as ‘dicks’. These agents were of
low importance compared with sulfur mustard and are considered obsolete.
1.4.4 Nerve Agents

1.4.4.1 Tabun and DFP
Tabun (GA), O-ethyl N,N-dimethyl phosphoramidocyanidate, was the first
nerve agent to be weaponised following its discovery by Schrader in Ger-
many in 1936.22 It evolved from structure–activity studies of organophos-
phates related to O,O-diethyl fluorophosphate (diethyl phosphorofluoridate)
(Scheme 1.3), whose toxicity had been reported some years earlier.60 A fea-
ture of these organophosphates was a displaceable ‘leaving group’ (F, CN)
on phosphorus, later shown to be displaced by a covalent reaction with a

of diethyl fluorophosphate, tabun and DFP.
serine hydroxyl group in the active site of the enzyme acetylcholinesterase.
By the end of WWII 12 000 tonnes of tabun had been produced in a plant at
Dyhernfurth, which was later dismantled and reconstructed in Russia. The
USA produced small stocks of tabun. The first known use of tabun was more
than 40 years later by Iraq in the conflict with Iran.34
Tabun is the easiest of the nerve agents to produce, essentially by a two
to three stage process from industrially available chemicals.51–54 It has less
favourable physicochemical properties than the other weaponised nerve
agents; its vapour pressure is quite low (bp 248 °C) and it is the least stable
towards moisture in the environment. Added to this, its lower inhalation tox-
icity compared with sarin and soman, and its much lower percutaneous tox-
icity compared with VX, it is regarded as being obsolete as a military agent.
Its ease of synthesis might make it attractive to proliferators with a limited
chemical industry or to terrorists.
UK and US chemists were less successful in developing a nerve agent
during WWII. The primary candidate was DFP (Scheme 1.3), studied by
Saunders and colleagues at Cambridge University.24,61 DFP had toxicity
approximately one fifth to one tenth that of sarin, with volatility closer to
soman. Its only advantage over sarin was ease of synthesis.
1.4.4.2 Sarin, Soman, Cyclosarin and 2-Methyl GF

Further molecular modification of tabun-like compounds in Schrader’s lab-
oratory produced sarin (named after the team that discovered it, Schrader
Ambros Rüdriger and Van der Linde). Sarin (GB), O-isopropyl methylphos-
phonofluoridate (bp 150 °C), is more volatile and more potent than tabun
(Scheme 1.4). It was produced in Germany on a pilot plant scale. Schrader sub-
sequently developed the less volatile soman (GD), O-pinacolyl methylphos-
phonofluoridate (bp 198 °C). Soman was later to achieve high importance
because of the resistance of exposed animals to medical countermeasures
(specifically the resistance of inhibited acetylcholinesterase to reactivation
with oximes). Soman became a leading candidate for weaponisation in the
USA, and was weaponised on a moderate scale in Russia, neat and thickened.
After disclosure of the German nerve agent programme, chemists from sev-
eral nations pursued further structure–activity studies over the next 20 years.
One avenue explored was analogues of sarin and soman with intermediate
volatility, i.e. between that of soman and tabun. Such agents (IVAs) could pen-
etrate semi-permeable protective clothing, and present both an inhalation

of sarin, soman, GF and 2-methyl GF.
18 Chapter 1
and contact hazard. Leading candidates in the US programme were cyclosarin
(GF), O-cyclohexyl methylphosphonofluoridate (bp 239 °C), and 2-methyl GF,
O-2-methylcyclohexyl phosphonofluoridate (bp 247 °C; Scheme 1.4). Neither
is known to have been weaponised during this period, although a mixture of
cyclosarin and sarin was used in crude binary form by Iraq in the 1980s.
1.4.4.3 V Agents
In the 1940s it was recognised that nerve agents act by binding to and inhib-
iting the enzyme acetylcholinesterase.62 This enzyme is the body’s mecha-
nism for inactivating the neurotransmitter acetylcholine. Inhibition of the
enzyme causes an excess of acetylcholine at nerve junctions and choliner-
gic neurones, producing excessive stimulation of cholinergic receptors. A
logical avenue to increase affinity for the enzyme was to explore pesticide
or nerve agent analogues with a structural feature that mimicked the natu-
ral neurotransmitter. Tammelin and co-workers63,64 in Sweden published a
series of papers on some highly toxic compounds known as Tammelin esters
(Scheme 1.5), but these were solids and had poor stability.
At the same time, chemists in the plant protection laboratories of ICI in
the UK were studying systemic pesticides with such features. This research
produced amiton, which possessed unusually high percutaneous toxicity.26
Modification of amiton in a UK/US/Canada military collaboration led to the
development of the V series of nerve agents, characterised by low volatility,
high percutaneous toxicity and high systemic toxicity. The analogue O-ethyl
S-(2-diisopropylaminoethyl) methylphosphonothiolate, given the military
designator VX, was assessed as possessing the optimum combination of
toxicity and storage stability, and was later produced and weaponised by
the USA. Chemists in Russia independently discovered the same series of
compounds, eventually leading to the weaponisation of a close analogue of
VX, O-isobutyl S-(2-diethylaminoethyl) methylphosphonothiolate, known as
R-33, RVX or VR (Scheme 1.6).
Of the three types of weaponised nerve agents, sarin became the primary
non-persistent agent of modern arsenals, and VX or RVX the primary per-
sistent agent together with sulfur mustard. Tabun was the least effective
nerve agent and gradually became redundant.
1.4.4.4 Other Nerve Agents

Two additional series of nerve agents are worthy of mention. Research on
IVAs in several countries led to the analogue known as GV, O-(2-dimethylami-
noethyl) N,N-dimethyl phosphoramidofluoridate (Scheme 1.7). The name GV
was coined by Czech chemists to indicate properties of both G and V agents.65
The US military designator was GP. GV is a hybrid structure incorporating
structural features of tabun, sarin and V agent. GV had true intermediate
volatility properties (bp 226 °C, volatility 527 mg m−3 at 25 °C),66 producing
sufficient vapour to cause an inhalation hazard, and possessing percuta-
neous toxicity approaching that of the V agents. GV might have become an

of amiton and a Tammelin ester.

of VX and RVX.
Scheme 1.7 Structure

of GV.
important threat agent had it not had very poor storage stability. It has been
suggested that a binary version might be feasible.65
In recent years, there has been much speculation that a fourth generation
of nerve agents, ‘Novichoks’ (newcomer), was developed in Russia, begin-
ning in the 1970s as part of the ‘Foliant’ programme, with the aim of finding
agents that would compromise defensive countermeasures.67,68 Information
on these compounds has been sparse in the public domain,30,68–70 mostly
originating from a dissident Russian military chemist, Vil Mirzayanov.69 No
independent confirmation of the structures or the properties of such com-
pounds has been published.
1.4.5 Riot Control Agents

RCAs are peripheral chemosensory irritants that target the eyes, airways
and/or skin.71,72 The 1997 CWC defines them as ‘Any chemical not listed
in a Schedule, which can produce rapidly in humans sensory irritation or
disabling physical effects which disappear within a short time following
termination of exposure’.1 Use for riot control purposes is permitted under
the CWC, but not for military harassment, and stocks of RCAs must be
declared. Most of the major RCAs [CN, CS and dibenz[b,f]-1,4-oxazepine
(CR); Scheme 1.8] are low volatility solids and, unless they are used in solu-
tion in a spray, they need to be aerosolised for efficient use, for example
using pyrotechnic munitions or dispersed as micronised powders.
More than 20 eye irritants (lachrymators) and upper airway irritants were
used in WWI.73 Bromobenzyl cyanide (CA, BBC) emerged as the most effective
20 Chapter 1
lachrymator. DM became available shortly after WWI. Both CA and DM were
used for law enforcement and stockpiled as military harassing agents. CA
was soon replaced in the 1920s by the more effective and safer lachrymator
CN. CN in turn was largely superseded in the 1950s by the more potent and
safer CS, which has remained the RCA of choice in many countries. DM is
now considered too toxic to be used as a RCA. The most potent eye irritant
of the RCAs, CR, was developed in the UK in the 1960s following its chance
discovery in a university laboratory. CR has an exceptionally high safety ratio
but, because it is difficult to decontaminate and residues may persist for
long periods, it has rarely been used in civilian environments. Small stocks
were declared to the OPCW. More recently, pepper spray [oleoresin capsicum
(OC)], containing capsaicin and related irritants, or a synthetic analogue
of capsaicin such as nonivamide [pelargonic acid vanillylamide (PAVA)],
have been widely adopted as aids to arrest and in personal defence sprays.
One other irritant is worthy of mention: 1-methoxycycloheptatriene (CHT)
Scheme 1.8 is a volatile liquid (bp 115 °C) with powerful irritant action,
particularly on the eyes. Although easily disseminated as a vapour, CHT has
not been adopted because of toxicological concerns.74
1.4.6 Incapacitants
A search for military incapacitating agents began in the 1950s, and contin-
ued for at least four decades.32,75–77 Incapacitants were later sought for law
enforcement and counter-terrorism purposes, particularly after a spate of
aircraft hijackings and other hostage situations in the 1960s and 1970s.
This period saw a major expansion of the pharmaceutical industry, which
invested heavily in research centres seeking new drugs. Large numbers of
experimental drugs were studied in animals, in contrast to modern drug
research, which uses animals more sparingly. Particular advances were made
in drugs that affected the brain. Examples are morphine-like analgesics

of RCAs.
(opioids), various classes of intravenous anaesthetics, benzodiazepines (pre-
scribed and manufactured in very large quantities), dopamine antagonists
for the treatment of schizophrenia, and dopamine agonists for Parkinson’s
disease. Some of the dopamine agonists investigated, such as derivatives of
apomorphine, induced vomiting in dogs at microgram doses.27,78
An early candidate incapacitant in the USA was phencyclidine (sernyl, PCP;
Scheme 1.9), which was marketed as an intravenous anaesthetic but soon
withdrawn because of complex psychotomimetic effects. PCP has a sim-
ple structure and is exceptionally easy to synthesise and it became a major
drug of abuse in the USA. It was eventually discarded as a potential military
incapacitant because of its low potency and a tendency to induce unpredict-
able and sometimes violent behaviour. A related drug, ketamine, has more
recently been studied by Czech scientists as a possible incapacitant in admix-
ture with other depressant drugs.79 The hallucinogen LSD was extensively
researched as a disruptive military agent by the USA but was rejected because
of the unpredictability of its effects and cost of synthesis.77
The US military eventually selected the centrally and peripherally acting
cholinergic antagonist BZ (Scheme 1.9), and manufactured approximately 50
tonnes for weaponisation in the 1960s. Several other nations are suspected
to have weaponised small quantities of BZ or an analogue. BZ had a num-
ber of disadvantages, particularly its slow onset and long duration of action,
incapacitation in terms of the ability to conduct military operations was diffi-
cult to judge, and it was expensive to manufacture. It was eventually declared
obsolete by the USA in 1976 and destroyed in the late 1980s.77
During the 1960s, potent opioids were the major focus of incapacitant
research in several countries. An example is etorphine (M-99), a semi-syn-
thetic hexacyclic opioid that is approximately 1000 times more potent than
morphine and marketed for veterinary use as a knockdown agent for large
game animals. Some prodeine analogues were patented as military incapac-
itants.80 In the 1970s and 1980s attention moved to a structurally simpler
and totally synthetic class of opioids known as the fentanyls (Scheme 1.10).81
The parent drug fentanyl, initially a product of the Belgian drug company
Janssen Pharmaceutica, is one of the most widely used intravenous analge-
sic/anaesthetics, with potency in humans 50–100 times that of morphine.
Some of its analogues are up to 10 000 times more potent than morphine

of PCP, ketamine and BZ.
22 Chapter 1
and rank amongst the most potent drugs known. One such drug, carfentanil,
is also used to knock down large game animals for veterinary or conserva-
tion purposes. In 2002, Russian Special Forces used an aerosolised mixture
of two fentanyls, carfentanil and the clinically used short acting, fast onset
analogue remifentanil (Scheme 1.10), to end the siege of a Moscow theatre
by Chechen terrorists.43 Approximately 130 of the 800 hostages died in this
operation, a major factor being the inherent respiratory depressant activity
of morphine-like compounds. Other depressants that have attracted serious
attention as possible incapacitating agents include α-adrenergic agonists
such as medetomidine and benzodiazepines.82
Incapacitants have a somewhat ambiguous status under the CWC. Devel-
opment and use is permitted for ‘law enforcement purposes’. However,
neither of the terms incapacitant and law enforcement are defined by the
Convention, and this could become a grey area, for example in the context
of peace keeping operations. Much concern has been expressed that this
ambiguity would allow development of agents that clearly have dual use
potential, military as well as law enforcement.83
1.4.7 Future Developments

Predicting the future of CW has proved notoriously difficult. Although many
hundreds of compounds have been assessed for CW potential, in defensive as
well as offensive programmes, the threat with regard to agents has remained
largely unchanged over the past 40 years. Sulfur mustard and G- and V-type
nerve agents have remained the most important threats. These agents are
relatively easy to manufacture, and they have close to the ideal physicochemi-
cal properties required of an agent. The most notable developments over this
period have been the proliferation of such agents to other nations, particu-
larly in the Middle East, use by terrorists in Japan, and the emergence and
use of fentanyl analogues as incapacitants.
Advances in the life sciences are accelerating at an unprecedented rate,
and concern has been widely expressed that some developments might pose
a challenge to the CWC if they were seriously applied to the development of
new agents.84 Examples are accelerated drug discovery (e.g. parallel synthe-
sis with high throughput screening), advances in neuroscience (leading to

of fentanyl and two analogues.
85
new incapacitants, e.g. derived from bioregulators), and the growing con-
vergence of chemistry and biology (e.g. synthetic biology, bio-production).86
Some of these concerns are arguably being overstated. The Scientific Advi-
sory Board of the OPCW, in its last five year report to the Director General on
advances in science and technology,87 noted these developments and others
as warranting surveillance and periodic review, but did not see any near term
threat to the Convention. It is pertinent to note that the most modern CW
agents known to have been weaponised, the V agents and BZ, are products of
1950s research, and the fentanyls used as incapacitants in Moscow are prod-
ucts of classical drug research undertaken in the 1960s to 1980s.
Although the CWC specifies certain categories of CW agent and their
precursors for declaration and verification purposes (in the Annex on
Chemicals), any emerging agents would still be captured under what is
unofficially referred to as the ‘general purpose criterion’. Article II states
‘For the purposes of the Convention, Chemical Weapons means... toxic
chemicals and their precursors, except where intended for purposes not
prohibited under this convention, as long as the type and quantities are
consistent with such purposes...’.1 It is therefore to be hoped that the near
universality of the CWC, together with ever more sophisticated means of
surveillance and verification, will dissuade potential proliferators from
risking the opprobrium and political or economic repercussions of contra-
vening the Convention.
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nerve gas in ’91, Baltimore Sun, 16 Sept 1992, 3A.
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28 Chapter 1
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Chapter 2
Toxicology of Vesicants
John Jenner*a
a
Biomedical Sciences Department, Dstl Porton Down, Salisbury, SP4 0JQ UK
*E-mail: jjenner@dstl.gov.uk
2.1 Introduction
During the First World War (WWI) the need to produce casualties in troops
wearing respiratory protection drove the search for agents active through, or
on, the skin. The first skin damaging agent used was sulfur mustard (SM), a
vesicant, so called because of its ability to produce blisters. SM changed the
number and type of casualties produced by chemical weapons. Prior to its
use comparatively few casualties reported to aid stations since many died
in the field and many of those who did report for medical assistance were
returned to the lines, fit for duty. SM produced many more disabled casual-
ties who spent several weeks or months in hospital before being returned to
duty and many were sent home requiring long periods of convalescence. A
similar experience was recorded between 1982 and 1989 in Iran. Towards the
end of WWI Winford Lee Lewis rediscovered and purified an arsenical based
vesicant,1 Lewisite (L), that combined the lethal effects of the earlier inhaled
chemical warfare agents (CWAs) with the skin damaging effects of SM. There
is no reliable record that L was ever used in warfare but it was manufactured
by Germany and Japan, and has also been mixed with SM to produce a mix-
ture with a lower freezing point than SM alone.

29
30 Chapter 2
In the years between WWI and WWII other mustards were experimented
with and the nitrogen mustards showed some effectiveness as vesicant CWAs,
although they were never weaponised.
This chapter describes the toxicology of the vesicants. There is a large
amount of literature on the toxicology of these agents and any account must
be selective. For additional accounts the reader is referred to one of the many
authoritative reviews published over the past 20 years.2–7
Many of the reports of studies carried out in the early years of vesicant
research lack certain pieces of vital information or were poorly designed by
modern standards. In many reviews these studies are excluded for under-
standable reasons. These reports are reviewed here and their short comings
highlighted, because they contain valuable information and some of the
observations made are not reported elsewhere.
2.2 Sulfur Mustard

SM, commonly known as “mustard gas”, was one of the first chemicals used
in modern warfare. Since its use on the battlefield of Ypres in 1917 it has
been used for little else but to wage war. In civil use it found brief employ-
ment as an anticancer agent during the 1960s, and has been used at low
concentrations in creams used to treat psoriasis, but its association with
the production of cancer terminated its use in medicine. With the forma-
tion of the Organisation for the Prohibition of Chemical Weapons (OPCW)
to enforce the international Chemical Weapons Convention (CWC), SM
is only likely to be encountered in dealings with non-compliant nations,
anti-terrorist operations, during demilitarisation operations or in defence
research.
2.2.1 Mechanism of Action

In the 90 years since SM was first used in warfare much research has been
done to identify how it interacts with cells to produce the biological effects
described below. The result has been exhaustive literature on its effects on
cellular biochemistry, but the primary biochemical lesion, or lesions, leading
to the tissue damage that SM causes remains unknown.
2.2.1.1 Chemical Reactivity
In aqueous solution the β-chloroethyl functional group of SM forms a reactive
cyclic intermediate, an episulphonium ion (ESI),8–11 that subsequently reacts
with a nucleophile to form an alkylated product. This is a nucleophilic substi-
tution (SN) reaction. If the rate determining step in the reaction is unimolecu-
lar these reactions are denoted as SN1, if bimolecular, SN2 (Scheme 2.1).
An alternative mechanism of alkylation involves the formation of a highly
reactive carbonium ion intermediate (Scheme 2.2).12
Toxicology of Vesicants 31
Scheme 2.1
Scheme 2.2
The reactive intermediate then reacts with a number of tissue constituents

as described in the following sections. Since SM has two chloroethyl groups
it can react with two nucleophiles to form cross-links within and between
macromolecules, which is presumed to be the reason for its toxicity.
2.2.1.2 Alterations to DNA
SM alkylates guanine (G) residues that then form base pairs with thymine (T)
rather than with cytosine (C) residues, resulting in coding errors and inaccu-
rate protein synthesis. Damaged G residues can be excised from the mole-
cule causing damage such as the loss of promoter regions and stop codons.2
Cross linking of pairs of G residues produces the most cytotoxic lesion, caus-
ing total disruption of metabolic cellular function.6
Approximately 75% of DNA alkylations are monofunctional and the
cross-linking alkylations account for only 25% of DNA alkylations; the rel-
ative proportion of these lesions in DNA is constant, and the total number
of alkylations in DNA has a linear relationship with SM dose.13 The ratio of
inter- to intra-strand cross-links is 1 : 2.14,15 The significance of inter-strand
cross-links between double helix DNA strands is that cell division ceases
because DNA polymerase is ineffective on such a structure; the cross-linking
lesions are approximately 10-fold more toxic to the cell than is the case for
the monofunctional adducts of SM.16 The effect of SM on rapidly dividing
cells is particularly severe (notably in the gut and bone marrow).2
The main product of DNA/RNA alkylation by SM is the N7 nitrogen in
guanine, resulting in the mono adduct (7-(2′-hydroxyethylthioethyl)-gua-
nine), and the relatively minor inter- and intra-strand di-adduct di-(2-
guanin-7′-yl-ethyl) sulphide, as well as the DNA N3 nitrogen of adenine
(3-(2′-hydroxyethylthioethyl)-adenine).16–19 Traces of an O6 guanine adduct
(O6-(2′-hydroxyethylthioethyl)-guanine) alkylation product have also been
reported.20 There is some evidence that damage to DNA is dependent on the
mitotic state of the cell, so that the cell’s DNA has a differential sensitivity to
SM depending on what part of the cell cycle it has entered.21–23
32 Chapter 2
Several hypotheses have been proposed to explain how the alkylation of
cellular constituents might cause the pathology produced by SM. Papirmeis-
ter et al.24 proposed the involvement of reduced NAD+. The “Papirmeister
Hypothesis” states that:

a. SM alkylates purines in DNA (i.e. G and A)
b. Backbone breaks are produced by apurinic endonucleases at these
sites
c. The chromosomal enzyme poly(ADP ribose) polymerase (PARP) is acti-
vated (an enzyme involved in DNA repair)
d. PARP uses NAD+ as a substrate and depletes cellular NAD+
e. Glycolysis is inhibited, resulting in the disturbance of energy
metabolism
f. Cell death

However, NAD+ depletion is not the only mechanism involved in cell death.
Prophylactic or therapeutic treatment of human epidermal cell cultures with
NAD+ precursors (i.e. nicotinamide) in an attempt to maintain NAD+ levels
do not protect these cells from SM.25
Other mechanisms of cell death have been described,26 in which the ini-
tiating step is a lowering of intracellular protein thiol levels caused either
by a direct reaction with SM or by depletion of glutathione (GSH). GSH reg-
ulates the equilibrium state of the intracellular pool of exchangeable Ca2+,
most notably the endoplasmic reticulum Ca2+ sequestering mechanism,26 by
prevention of the oxidation of thiols, which is essential for the activity of Ca2+
ATPase.
2.2.1.3 Alterations to Other Cellular Components and Processes

There is evidence of SM alkylation of cellular proteins in vitro27–32 as well as
loss of important enzyme cofactors such as GSH,33 and the modification of a
number of biochemical processes such as the activation of proteases34,35 and
the loss of energy storage molecules such as ATP36 and NAD.37 Although the
eventual pathological effect of SM exposure is tissue necrosis, there is evi-
dence of the activation of apoptosis,38,39 the loss of calcium regulation,40 the
loss of cell cycle regulation,41 and perturbation of the cytoskeleton,29 cyto-
kine production42,43 and basement membrane components.44 This suggests
that many cellular pathways contribute to SM-induced tissue pathology.
2.2.2 Toxicokinetics, Metabolism and Distribution

SM is a lipophilic compound that is absorbed readily by the skin, eyes, respi-
ratory tract and gut. There is a substantial literature database for SM kinetics
in animals, as well as some data obtained from either accidental or deliber-
ate human exposures. There appear to be differences in the pattern of distri-
bution of SM between humans and other animals, although whether this is
a true difference or the result of variations in measurement methods, time
since exposure or route of exposure is uncertain.45
SM is rapidly absorbed from the site of administration in all species,
whereupon it either reacts rapidly with macromolecules (alkylation) or is
metabolised, the major metabolites being thiodiglycol and GSH adducts.
Toxicologically relevant doses of SM rapidly penetrate the surface layers
of the skin (stratum corneum) and there is evidence that a depot is formed
in the skin during absorption, although the location and toxicological rele-
vance of this depot are not certain.
2.2.2.1 Studies in Animals
2.2.2.1.1 Dermal. Cullumbine46 used a histological stain that formed an
insoluble black complex with free SM, but not with degradation products
such as thiodiglycol, to show “free” unreacted SM in the skin at 15 min post
exposure but not at 30 min, leading to the conclusion that “free SM, after
penetration, did not exist in the skin except in the epidermis”. Using micro-
autoradiography, Axelrod and Hamilton47 showed that label was present
within both the epidermis and the dermis, although whether the label
detected was free SM or a degradation product was not determined.
When 35S-labelled SM was applied to the skin of rats under occlusion, >90%
of the applied dose was absorbed within 6 hours.48 Uptake increased linearly
with the applied contamination density in the range of 3–605 µg cm−2, reach-
ing a maximum of approximately 7 µg cm−2 min−1 at 955 µg cm−2. After 6
hours, approximately 75% of the applied radioactivity had passed through
the skin and distributed systemically, 25% was retained in the skin, up to
30% was excreted in the urine and 5–8% remained in the blood. The half life
of the radioactivity in the plasma was 2.4 days, but detectable radioactivity
remained bound to haemoglobin 40 days after application.
Measurement of radiolabel cannot distinguish between parent com-
pounds and metabolites, so no conclusions can be drawn about the identity
of distributed and excreted material from these studies alone.
2.2.2.1.2 Inhalation. SM is readily absorbed across the respiratory tract,

including across the nasal membrane.49 Langenberg et al.50 showed that
following inhalation in the guinea pig, the blood mustard concentration
reached a maximum concentration of 300 mg m−3 5 min after exposure.
The appearance of DNA adducts in tissues 4 hours after exposure to lower
concentrations (160 mg m−3 for 5 min) showed that the mustard distributed
widely to the lung, spleen, bone marrow, nasal epithelium, nasopharynx, larynx
and carina.
2.2.2.1.3 Other Routes. Radioactivity appears in the kidney, lung and liver
after intravenous administration of radiolabelled SM to the rabbit.51 Lower
levels of radioactivity were also found in the bone marrow, spleen, stomach,
duodenum, brain, heart, muscle, skin and thyroid gland. In the guinea pig,
34 Chapter 2
radiolabelled mustard likewise distributes widely, with label being found in
the bone marrow, spleen, blood and lung.50 The study showed a rapid distri-
bution phase, then slower elimination. Similar results were observed in the
rat after intravenous injection.52–54
Davison55 reported that after intravenous administration in the rat and
mouse, mustard is metabolised largely by conjugation with GSH, hydrolysis
and oxidation, with the major metabolites being GSH conjugates, thiodigly-
col conjugates and sulphones. Other authors have also reported cysteine
conjugates.56
Also after intraperitoneal (IP) injection in the rat, Black et al.57 identified
a large number of metabolites, confirming the earlier result of Davison55 as
well as identifying several other metabolites in the urine, nine of which were
characterised (Figure 2.1). Although some metabolites were the products of
initial hydrolysis, the majority resulted from combination with GSH and sub-
sequent metabolism to N-acetylcysteine conjugates or to methylthio/methyl-
sulphinyl derivatives by β-lyase. Each of these products could be oxidised to
the sulphoxide or sulphone. The proportion of the absorbed dose excreted
as thiodiglycol and thiodiglycol esters was only one tenth that shown by
Davison.55 The metabolite profile after IP injection57 was the same as after
cutaneous administration.48
2.2.2.2 Studies in Humans
2.2.2.2.1 Dermal. Renshaw† reported that >80% of SM applied to the skin
evaporates if the application site is left unoccluded,58 and the rate of pene-
tration of SM into human skin was 1–4 µg cm−2 min−1 from unspecified con-
tamination densities. Some authors claim that in humans the absorption
is via the sweat glands,59 but there is no empirical evidence to support this
hypothesis for SM.
2.2.2.2.2 Other Routes of Exposure. No systematic studies describe the

kinetics of SM following other routes of exposure in humans. In particular,
there are no reported studies of the kinetics of SM after inhalation or oral
exposure.
Significant amounts of thiodiglycol have been found in the urine of Iranian
soldiers exposed to SM during the Iran–Iraq war of the 1980s,60,61 although
the authors point out that low levels of this compound are also found in unex-
posed individuals. Unmetabolised SM was also found in both the urine and
faeces of Iranian SM casualties.62–64 Since these individuals were exposed on
†
In 1946 Renshaw published a review of work with SM carried out during WWII. This valuable
work, although classified when originally produced, has subsequently been released to the
public and the views expressed by Renshaw and his co-authors have had a formative effect on
our perception of SM toxicology over the past 50 years. The majority of the reports quoted in
Renshaw’s review have been lost or are inaccessible, and references to Renshaw’s conclusions,
although valuable, should be viewed in the light that his interpretation of the source material
cannot be confirmed.
Figure 2.1 Metabolic

breakdown of SM. Proposed metabolic fate of SM (adapted
from Black et al.57). Metabolites are 1: SM; 2: thiodiglycol; 3: thiodi
glycol sulphoxide; 4: mustard sulphoxide; 5: mono-GSH-SM ad
duct; 6: 1-[S-(N-acetylcysteinyl)]-2-(2-chloroethylsulphinyl)ethane; 7:
1-[S-(N-acetylcysteinyl)]-2-(2-chloroethylsulphonyl)ethane; 8: 1-[S-(N-
acetylcysteinyl)]-2-(ethenylsulphonyl)ethane; 9: di-GSH-SM adduct; 10:
1,1′-sulphonylbis[(2-S-cysteinyl)ethane]; 11: 1,1′-sulphonylbis[2-S-(N-
acetylcysteinyl)ethane]; 12: 1,1′-sulphonylbis(ethan-2-thiol) (putative
intermediate indicated by square brackets); 13: 1,1′-sulphonyl-
bis[2-(methylthio)ethane]; 14: 1-methylsulphinyl-2-[2-(methylthio)eth-
ylsulphonyl]ethane; 15: 1,1′-sulphonylbis[2-(methylsulphinyl)ethane].
the battlefield, they are likely to have been subject to exposure from a range
of routes, although primarily the inhalation and dermal routes combined.
The appearance of thiodiglycol in urine, following an accidental human
dermal exposure to SM, has also been demonstrated by Jakubowski et al.65
Thiodiglycol was detected in the urine for up to 13 days after the exposure.
36 Chapter 2
2.2.2.3 In vitro Studies

Chilcott et al.66 reported the in vitro measurements of 35S-labelled SM pen-
etration through human skin (heat-separated epidermal membranes and
full thickness skin) using Franz type static diffusion cells at 30–32 °C. The
measurements were made after exposing the skin surface to pure SM liquid
(finite, 10 µl, and infinite, 20 µl, doses) under occluded and unoccluded con-
ditions and after exposure to saturated SM vapour.
The resultant data are consistent with the studies reviewed by Renshaw58
who reported a penetration range of 60–240 µg cm−2 h−1 measured using a
variety of in vitro techniques over a skin temperature range of 23–37 °C. Chil-
cott et al.66 reported the flux of 35S-SM from saturated vapour under occluded
conditions through heat-separated human epidermal membranes as 110 ±
75 µg cm−2 h−1, which compared well to the figure of 162 µg cm−2 h−1 derived
by Nagy et al.67 by calculating the difference between SM delivered to and
recovered from the application device used to expose the skin of human
volunteers.
2.2.3 Acute Toxicity

SM is acutely toxic by all routes of administration, causing widespread tissue
destruction and inflammation at the portals of entry (skin, lung and gastro-
intestinal tract) and a suppression of all rapidly dividing cell populations,
such as bone marrow, epidermis and seminiferous tubules. After inhalation
exposure the lining of the respiratory tract is destroyed leading to pneumo-
nitis, which can be severe, with the complication of the formation of pseu-
domembranes that slough off and block the airways. Exposure of the skin
produces a delayed irritant response resulting in widespread tissue destruc-
tion similar to a thermal “burn”. SM can also penetrate the skin to produce
systemic toxicity related to its effects on rapidly dividing cell populations,
and bone marrow suppression is demonstrated by leukopenia, thrombocy-
topenia and anaemia. Dose response data are very limited, but there are suf-
ficient data to allow some estimates of the lethal dose by some routes to be
made (see below).
Humans exposed by inhalation for a short time to high concentrations of
SM go on to develop a variety of long term respiratory effects. There is a clear
relationship between exposure dose and the severity of subsequent effects of
SM, but no evidence to suggest that the severity of the long term effects bears
any relationship to the severity of the acute response.
2.2.3.1.1 Oral Exposure. There are few reports on the oral toxicity of SM.
In one study, the oral lethal dose, 50% (LD50) of SM [administered in polyeth-
ylene glycol (PEG) 300 or dimethyl sulfoxide (DMSO), respectively] in female
mice (groups of four) was 37 and 38 mg kg−1 after 7 days, and 8 and 10 mg kg−1
after 14 days. In male rats, the LD50 was 7 mg kg at 7 days, and 2 mg kg−1
68 −1
at 14 days (SM applied in PEG 300). The low number of animals limits the
usefulness of these estimates. The causes of death were not stated.
In a separate experiment, groups of mice were dosed orally with 4.83, 9.67
and 19.3 mg kg−1 SM in PEG and the histopathology was studied after 7 days.
Extensive DNA fragmentation in the liver was observed at the highest dose,
with a dose dependent decrease in GSH. There was dose dependent damage
to the liver, lungs and spleen. In the liver there was centrilobular necrosis
with occasional vacuolar degeneration of the midzonal cells, congestion and
haemorrhage. The lungs were congested and haemorrhagic with inflamma-
tion and neutrophil infiltration into the alveolar spaces. Bronchiole asso-
ciated lymphoid tissue showed granulovacuolar degeneration and spleens
were congested with an apparent increase in haematopoietic precursor cells.
2.2.3.1.2 Dermal Exposure. Since the first research efforts in 1917–1918,

a large range of animal species have been exposed to SM liquid and vapour
percutaneously. The species tested range from the standard laboratory spe-
cies (rats, rabbits, mice and guinea pigs) through to farm animals (such as
pigs), to the pack animals important in the early years of research (horses,
bullocks, donkeys and camels).69–71
Lethality. Dacre and Goldman72 reported the percutaneous LD50 of SM to

be 9 mg kg−1 in the rat, 92 mg kg−1 in the mouse, 20 mg kg−1 in the dog, ∼100
mg kg−1 in the rabbit, 20 mg kg−1 in the guinea pig and 50 mg kg−1 in the goat,
although they do not quote the source for these estimates or the time after
exposure when the estimates were made, and no details of the cause of death
are given (i.e. systemic toxicity or as a result of skin injury).
Vojvodic et al.73 estimated the percutaneous LD50 in rats using groups
of four or five animals. The percutaneous LD50 was 194 ± 4 mg kg−1 at 24
hours and 15 ± 4 mg kg−1 at 96 hours. Lethality was the only toxic endpoint
described in this study. A more recent study showed that in female mice
(groups of four) the percutaneous LD50 of SM (administered in PEG 300 and
DMSO, respectively) was 123 and 9 mg kg−1 after 7 days, and 5.7 and 5.0 mg
kg−1 after 14 days.68 In male rats the LD50 was 3 mg kg−1 at 7 days and 2 mg
kg−1 at 14 days (SM applied in PEG 300).68 In both of these studies either the
low number of animals used or the limited experimental details provided,
or both, limits the usefulness of the results. In both studies no details were
given of any measures taken to prevent ingestion of the material after percu-
taneous application, so the possibility of ingestion from the application site
cannot be excluded and the results should be interpreted in this light.
Sublethal Pathology. Venkateswaran et al.74 investigated the systemic

effects of sublethal percutaneous doses of SM (3.88, 7.75 or 15.5 mg kg−1 in
olive oil applied over a 1 cm2 area of close clipped skin) in groups of at least
six mice. The authors do not describe precautions to prevent ingestion or
the housing conditions of the animals after dosing. Animals were observed
38 Chapter 2
for 7 days and body weight gain declined in all groups compared with con-
trols after 3 days, falling below pre-dosing values in the highest dose group.
Weights of the spleen, liver and peripheral lymph nodes were significantly
reduced following exposure to the 7.75 and 15.5 mg kg−1 doses of SM, while
the adrenal weight was increased in all groups. There was dose dependent
damage to the thymus and spleen but no detectable decrease in peripheral
white blood cell (WBC) count, although there was a significant increase in
red blood cell (RBC) count and packed cell volume. The haematological,
spleen and thymus changes could be indirect consequences of local skin
damage (haemorrhage, oedema, dehydration, clinical shock, compensatory
erythropoiesis, etc.).
2.2.3.1.3 Inhalation
Pathology. An early study described the pathology of SM effects on the lungs
in detail.75 Three groups of dogs responded in different ways to whole body expo-
sure to SM vapour, although no details of the exposure concentrations or dura-
tions were given, but good detailed descriptions of the pathological response
were provided. The first group of animals died without extensive pneumonia
between 18 and 73 hours after exposure, the second group died with extensive
pneumonia 2–10 days after exposure and the third group survived. Animals
showed signs of mild irritation during exposure with lacrimation and some
increase in nasal and salivary secretion during longer exposures. At autopsy
the most noticeable feature was the formation of pseudomembranes covering
the interior surfaces of the tracheobronchial tree, consisting of necrotic epi-
thelium, cell debris, fibrin, leucocytes, mucus and, occasionally, RBCs.
A small number of animal studies have investigated the pathology asso-
ciated with exposure of the respiratory system to SM after intra-tracheal
administration.76,77 This method of administration eliminates effects on the
upper respiratory tract and is, therefore, more representative of inhalation
through the mouth in humans. A study by Anderson et al.76 examined groups
of four to seven anaesthetised rats exposed by intra-tracheal intubation to
vaporised SM (0.35 mg in 100 µl ethanol, 50 min). The pathology in animals
euthanised at 0, 1, 4, 6, 12, 18 and 24 hours post exposure was assessed and
a detailed histological and ultrastructural examination of the respiratory
tracts performed. Histological analysis showed little or no effect up to 4
hours post exposure, with lesions in the airways (trachea, bronchi and larger
bronchioles) developing after a latent period of 4–6 hours. Epithelial necrosis
and separation of the mucosal/submucosal interface began at 6 hours post
exposure. Isolated tracheal and bronchial epithelial necrosis and epithelial
sloughing were followed by formation of fibrinocellular pseudomembranes
within the airways 12–18 hours post exposure. Pseudomembranes were most
frequently associated with de-epithelialised areas overlying the bronchiolar
associated lymphoid tissue. Peribronchiolar and perivascular oedema were
present at 24 hours post exposure; at this time the alveoli appeared relatively
unaffected, containing little cellular debris and few inflammatory cells.
Anaesthetised large white pigs given inhaled doses of 60, 100 and 150 µg
kg−1 over 10 min, via an endotracheal tube to bypass the upper respiratory
tract, developed oedema of the tracheal lamina propria with inflammatory
cell infiltration and, in the high dose group, localised sloughing of the epithe-
lium by 6 hours post exposure.78 Damage in the lower lung was minimal and
the lung wet weight to body weight ratio was unchanged when the animals
were killed 6 hours after exposure. There was a dose dependent increase in
interleukins 1β and 8 in terminal bronchoalveolar lavage fluid and a decrease
in arterial oxygenation with an increase in shunt fraction, indicating some
damage at the gas exchange level.
The differences in the onset times of various pathologies in different ani-
mal studies are probably due to the differences in doses and susceptibility of
different species.
Lethality by Inhalation. There are very few quantitative determinations of the

lethality of SM after inhalation exposure. Box and Cullumbine79 described the
lethality of SM in mice after a single 10 min exposure to SM vapour (Figure 2.2).
The majority of animals died within 11 days but a number of later deaths were
observed in the groups receiving lower doses. The estimated 10 min LC50 was
101 mg m−3 (88–115; 95% CL) with a probit slope of 4.11 (3.09–5.14; 95% CL).
In a multi-species study of the lethality of inhaled SM, Cameron and Short49
investigated the inhalation lethality of SM in mice, rats, guinea pigs, rabbits
and goats using a closed system where the agent concentration decreased
during the exposure. The data from this study were not robust enough to sup-
port probit analysis for any species, but comparison of the mouse data with
the estimated dose response curve from the Box and Cullumbine study shows
Figure 2.2 Lethality

in mice. Dose response curve for lethality of inhaled SM in
mice for a 10 min exposure in a constant flow apparatus. Groups of
30 mice were housed individually during exposure and observed for 26
days.79 The data are shown with the line of best fit and 95% CL calcu-
lated from the data marked by crosses using the Finney method.84
40 Chapter 2
Figure 2.3 Lethality

of SM in mice: a comparison of percentage lethality. Compari-
son of the percentage lethality determinations in mice by Cameron and
Short49 with the dose response curve calculated from Box and Cullum-
bine’s79 study (±95% CL as in Figure 2.2). Symbols represent different
exposure times: 5 min (pluses); 10 min (triangles); 20 min (diamonds);
and 30 min (circles). The data are shown with the line of best fit and 95%
CL calculated from the data marked by crosses using Finney method.84
Table 2.1 Inhalation

toxicity of SM in rodents80.
Time (min) Mouse LCt50 (mg min m−3) Rat LCt50 (mg min m−3)
2 860 840
60 1380 990
360 4140 1512
that the mice in the Cameron and Short study were apparently more sensitive
(Figure 2.3). For species other than the mouse, too few animals were used in
each group to allow a meaningful analysis.49
In a later study McNamara et al.80 reported the lethal concentration, 50%
(LCt50) values for 2, 60 and 360 min exposures (Table 2.1) and stated that the
effect of the same dose of SM was less when delivered over a longer period of
time, which they concluded was due to biological detoxification of the agent.
However information on how this study was conducted is not reported in
any detail and the results should be evaluated in this light. A summary of the
toxicity studies in animals is given in Table 2.2.
2.2.3.2 Exposures of Humans
2.2.3.2.1 Dermal. The acute dermal effects of SM are a primary irritancy
reaction and are covered in detail in Section 2.4.
2.2.3.2.2 Inhalation. There are no non-anecdotal reports of acute effects

in humans by the inhalation route. The following is an account of the long
term effects of acute exposure.
Table 2.2 Summary
of acute toxicity studies in animals.
Dose or exposure Exposure Effective dose, LOAEL or
Study system concentration Exposure route time Effect NOAEL Reference
Anaesthetised 0.35 mg in 100 µl SM vapour 50 min Histological damage 76
male rats ethanol given to (intra- from 4–6 hours
(250–300 g) each animal tracheal) Ultrastructural effects
from 6 hours
Male guinea 0.3 ml kg−1 Bolus (intra- Severe lesions to tra- 0.5 × LD50 81
pigs (250– tracheal) cheal epithelium
300 g) from 5 hours
Dogs Unknown Whole body Pseudo-membrane 75
SM vapour formation
Mice 67–505 mg m−3 SM vapour 10 min Lethality Estimated LCt50 = 1010 mg min 79
m−3
Male guinea 0.73 mg kg−1 (admin- Aerosolised Early asthma like 1 × LD50
pigs (400– istered in 0.5 ml of SM (intra- symptoms
600 g) phosphate buffered tracheal) Later ARDS like signs
saline)
Male hairless 160 mg m−3 Nose only to 5 min Upper respiratory tract 1 × LCt50 = 800 mg min m−3 82
guinea pigs SM vapour damage
(400–500 g)
Mice (albino) 8.5, 16.9, 21.3, 26.8, Head only to 60 min Reduced respiratory NOAEL = 510 mg min m−3 83
42.3 and 84.7 mg m−3 SM vapour rate
Reduced body weight LOAEL = 1014 mg min m−3
14 day LCt50 = 2550 mg min m−3
Rats Not given Subcutaneous N/A Lethality LD50 (24 hours) 150 ± 12 mg 73
injection kg−1
LD50 (96 hours) 8 ± 3 mg kg−1
Anaesthetised Not given Intravenous N/A Lethality LD50 8.2 mg kg−1 (7.1–8.8; 95% 50
guinea pigs CL)
Rats Not given Percutaneous Lethality LD50 (24 hours) 194 ± 4 mg kg−1 73
LD50 (96 hours) 15 ± 4 mg kg−1
41
42 Chapter 2
Humans exposed during war are usually subject to a single acute expo-
sure of SM by inhalation, although exposure more than once and to more
than one chemical is possible, and such individuals may be included in the
studies reviewed. Those exposed during manufacture may have been repeat-
edly exposed to lower than lethal or incapacitating doses over a prolonged
period of time. Individuals may have been exposed to more than one chemi-
cal and there is no indication of the concentrations to which individuals were
exposed or for how long in any of these studies. This section reviews reports
of deliberate use of SM in warfare since those individuals examined are more
likely to have received a single acute exposure. Occupational (manufactur-
ing) exposures are reviewed in Section 2.8.2 under carcinogenicity.
First World War. In a 1922, a clinical study of 83 “pensioners” with rec-

ognised disability due to gas poisoning during WWI was performed. Some
of these people could have been exposed to more than gas and, although the
reports do not specify the gas to which they were exposed, it has been his-
torically assumed that the long term lung effects were due to SM. The prin-
cipal symptom was shortness of breath, but persistent cough, expectoration
and chest tightness were also common.85 Signs of emphysema were evident
in 26%, whilst another 20% had signs of chronic bronchitis. Similar effects
were also seen in a group of 2000 WWI US veterans.86
Second World War. There were no battlefield exposures to SM during com-

bat in WWII. However, an example of the effects of an acute exposure of men
to liquid SM that caused systemic poisoning occurred at Bari Harbour in
1943 when a ship carrying SM was bombed.87 Six hundred and seventeen
men were exposed to SM (mixed with petrol and oil), released onto the sur-
face of the sea, when they escaped from damaged vessels into the water.
Briefly, extensive eye damage, skin burns, respiratory tract damage and sys-
temic absorption occurred; the mortality rate was 13.6%. Casualties were
initially treated for shock and other injuries, and the SM containing oil was
not washed from their skin, since the presence of SM was not suspected.
Casualties developed eye effects within a few hours that became pronounced
the following morning. Casualties were in shock, thought to be from expo-
sure and immersion, but many of the casualties with severe hypotension did
not exhibit the clinical picture of surgical or medical shock or obvious signs
of SM poisoning. Very low arterial pressures were not associated with rest-
lessness or distress and the extremities were warm. Some of these effects
may have been due to loss of fluid into the skin as the burns developed, but
in the early stages when this effect was observed, there was little evidence of
skin burns. Loss of fluid into the tissues resulted in haemoconcentration and
the WBC count fell on the third or fourth day to 100 cells cm−3 or lower. At
autopsy the pathology indicated changes in the liver, haematopoietic system,
gastrointestinal tract and genitourinary tract consistent with poisoning by a
radiomimetic alkylating agent.
Iran–Iraq War. Over 100 000 medical casualties were reported from the
Iran–Iraq War with symptoms related to SM exposure. Of 61 victims of SM
injury between the ages of 15 and 30 years described 2–4 weeks following
exposure,88 21 were followed for 15 months with X-ray and spirometry inves-
tigations. All had persistent cough, 75% excessive sputum production and
62% shortness of breath. One subject showed improvement during the fol-
low up period and 20% of the group appeared to have a “normal” pattern
of respiratory function. Most studies of the long term pulmonary complica-
tions in SM victims for this conflict have used small sample sizes; the excep-
tions to this are reviewed below. In addition, none of the studies have explicit
information on exposure dose or route making interpretation difficult.
In a study of 1337 soldiers89 the Cumulative Incidence Rate of pulmonary
complications was 31.6% with the lowest annual incidence rate noted during
the first year of follow up and the highest rate recorded in the seventh year.
In 220 male patients surveyed 6–13 years after exposure,90 all patients had
at least one complaint such as chronic intractable cough, dyspnoea, suffo-
cation or haemoptysis. Bilateral wheezing and wet rales were common. Of
40 male Iranians surveyed 16–20 years after exposure 100% complained of
coughing, 95% sputum production, 85% dyspnoea and 60% haemoptysis.91
The main respiratory complications were chronic obstructive pulmonary dis-
ease (COPD) in 35%, bronchiectasis in 32.5%, asthma in 25%, large airway
narrowing in 15%, pulmonary fibrosis in 7.5% and simple chronic bronchi-
tis in 5% of patients.
A group of children and teenagers exposed at Halabja in 1988 were admit-
ted to hospital some 18–24 hours post exposure, along with adults from the
same incident.92 The onset time of manifestations in children and teenag-
ers was earlier than in adults (4–18 hours in children versus 8–24 hours in
adults); first symptoms were cough and vomiting in children, but not adults;
symptoms on the face and neck were dominant in children and involvement
of genitalia less frequent. The severity of ophthalmic manifestations was
greater in children as well as the frequency of pulmonary and gastrointesti-
nal symptoms. These results imply that children may be more sensitive than
adults, but the possibility that the differences are due to higher exposures
than in adults has not been addressed.
SM has been associated with bone marrow toxicity leading to pancytope-
nia; however, in a study of 318 patients 10 years after exposure, the average
RBC, haemoglobin, total WBC, neutrophil and lymphocyte counts were not
significantly different from controls.93 In another study, although the route
of exposure is not clear and patients may have been exposed by multiple
routes, the effects on the haematopoietic and immune systems have been
demonstrated.94 Initially there is leukopenia followed by thrombocytopenia
and anaemia, patients who survived had continued haematological changes,
with WBCs decreasing below 1000 cells cm−3. Bone marrow biopsies revealed
hypocellular marrow and cellular atrophy. Patients had decreased T-cells (54%
lower than normal), monocytes (65%), eosinophils (35%) and neutrophils
44 Chapter 2
(60%), while B-cells were increased up to 7 weeks post exposure. C3, C4,
CH50, IgG and IgM were all increased in response to the initial exposure up
to 6 months post exposure. Eight years post exposure patients showed an
increase in atypical leukocytes and expressed less CD56 [a natural killer (NK)
cell surface marker]. After 14 years, casualties reported three major prob-
lems: recurrent infections leading to septicaemia, respiratory difficulties and
lung fibrosis. Acute myeloid leukaemia was increased 18-fold and lympho-
blastic leukaemia 12-fold, possibly due to a shift from a Th1 response to Th2
response, but this is only briefly reported without giving a total number of
casualties or cases of leukaemia.94 After 16–20 years there were significant
increases in WBC and RBC counts, α1, α2 and β globulins.95 Monocytes and
CD3+ lymphocytes were increased and CD16+ CD56+ cells were significantly
lower. Transforming growth factor beta (TGF-β) levels are also increased and
could be responsible for promoting fibrosis in lung tissue.96 This increase in
fibrous tissue and therefore a decrease in lung function could be indirectly
responsible for the increase in RBC number and haematocrit.
2.2.4 Irritation and Corrosiveness

Is SM an irritant or a corrosive? The Chemicals (Hazard Information and
Packaging for Supply) Regulations (2009) define corrosives as “substances
and preparations which may on contact with living tissues, destroy them”
and irritant chemicals as “non-corrosive substances which, through immedi-
ate, prolonged or repeated contact with the skin or mucous membrane, may
cause inflammation”.‡ SM does not destroy tissues on immediate contact,
but produces an inflammatory response that results in tissue destruction. As
such, SM is an irritant, although it can produce injuries of such severity that
they are similar to those produced by a corrosive.
2.2.4.1.1 Dermal. Early studies97 described similar pathology of liquid SM
on the skins of rabbits, guinea pigs and cats. Within 2 hours of application
of a “0.002 cc” droplet there was marked oedema over a larger area than the
spread of the drop. The oedema was subcutaneous with a defined edge. After
3 days the area was described as “necrotic” and the surface “sloughs” without
forming blisters, the slough being held in place by the fur. Over the follow-
ing 3–4 weeks the lesion gradually elevated, contracted and was cast off to
reveal healed skin beneath. The authors commented on the persistence of
the lesion and the slow healing in the absence of infection.
Similar histopathological changes have been observed in mice98 and
Yucatan mini-pigs.98 Pig skin is histologically and biochemically similar
to that of humans and the pathology of SM injury is also similar in time
‡
Available as a UK Statute from the UK National Archive at http://www.legislation.gov.uk/
uksi/2009/716/pdfs/uksi_20090716_en.pdf
course and character. In Yucatan mini-pigs only minor changes (focal vacu-
olation, heterochromatin condensation, swelling of the endoplasmic retic-
ulum and some migration of polymorphs from capillaries in the superficial
dermis) were evident in the skin 2 hours after exposure to saturated vapour,
under occlusion, for 6 hours. At 6 hours the endothelial cells of the papil-
lary dermal capillaries appeared swollen with some congestion of the lumen.
This pathology became progressively more pronounced over the following
18 hours and at 24 hours the papillary dermal collagen appeared coagulated
and hypereosinophilic, and no fine structure could be discerned in the capil-
laries, which were severely congested. The melanocytes in this species showed
effects before the surrounding epidermal cells, although the effects were the
same as in keratinocytes, i.e. vacuolation, nuclear condensation, etc. Similar
pathological changes were also observed in the larger strains of pig.99,100
A more detailed study101 showed similar effects in guinea pigs and rabbits
treated with doses between 25 and 250 µg cm−2 applied to the skin in meth-
ylene chloride. These authors described the time course of lesion develop-
ment and healing, which was similar in both species over the entire dose
range (Table 2.3).
Studies of the effects of 10 µl of SM, vaporised (8 min), or 2 µl of SM liquid
(30 min) applied to the skin of hairless guinea pigs102 revealed similar inju-
ries to those seen in the rabbit and furred guinea pig.101 At 12–24 hours the
basal and supra-basal epidermal cells showed extensive cytoplasmic vacuoli-
sation, swollen endoplasmic reticulum, nuclear pyknosis and cellular necro-
sis. Microblisters were evident.
2.2.4.1.2 Inhalation. Vijayaraghavan et al.83 investigated the effects of the

inhalation of SM on breathing patterns in groups of four mice exposed to
concentrations of 8.5, 16.9, 21.3, 26.8, 42.3 and 84.7 mg m−3 for 60 min, head
only in plethysmography tubes with acclimatisation and recovery periods.
These exposures equate to the concentration–time products (Cts) of between
510 and 5082 mg min m−3 and are within the lethal range. Animals exposed
to concentrations higher than 21.3 mg m−3 showed statistically significant
Table 2.3 Time

course of dermal effects of SM in guinea pigs and rabbits101.
Time Effect
30–60 min Erythema at site of application
1–4 hours Erythema enlarged
5–8 hours Oedema noticeable
12–18 hours Erythema and oedema extended well beyond the application site
24–48 hours Centre of the lesion appeared blanched with some petechial
bleeding
48–72 hours Epidermal necrosis covered by thin fibro-serous exudate wherever
SM had touched the skin
3–5 days Visible lesion plateau
5–10 days Erythema and oedema slowly subsided, serous exudate dried, scab
formed
10–14 days The scab sloughed revealing a thin, shiny layer of new epidermis
46 Chapter 2
concentration and time dependent decreases in respiratory rate after 15–20 min
of exposure. There was a reduction in body weight over the 7 days follow-
ing exposure that was statistically significant after exposure to 16.9 mg m−3
(Ct = 1014 mg min m−3). Exposure to 8.5 mg m−3 (Ct = 510 mg min m−3) pro-
duced no observable effect on respiratory rate at the time of exposure. It
should be noted that this author reported an estimated 14 day LCt50 of 2550
mg min m−3 in contrast to the 1010 mg min m−3 reported in earlier studies in
mice.79 The lack of effect on breathing patterns is indicative of no irritation
of the respiratory tract at these Cts.
2.2.4.1.3 Ocular. Warthin103 compared clinical descriptions of SM induced

eye lesions in humans with the pathology of experimental lesions in rabbits
and concluded that the injuries were very similar. The degree and latency
of the injuries were dose dependent, higher concentrations and/or longer
exposure times producing more severe injuries with a shorter latency. It was
also clear that injury to the cornea is critical to loss of sight or permanent
injury. Five “well recognised stages” of severe corneal damage are described
by Hughes:104

1. Immediate damage to the corneal epithelium with oedematous cloud-
ing and necrosis of the stroma;
2. After 5 hours, an infiltration of polymorphonuclear cells at the schlero-
corneal junction, extending into the corneal stroma;
3. At 5–7 days there is a clinical improvement of the opacity with dimin-
ished oedema of the stroma;
4. A progressive vascularisation of the cornea extending in from the lim-
bal vessels that may continue for several weeks;
5. Persistent ulceration of the cornea for weeks, or recurrent ulceration
after a latent period of years.

Kadar et al.105 developed a model for ocular lesions induced by SM
vapour in rabbits and showed that the clinical and pathological course
of SM injuries in this species was similar to those described in humans
[see Table 2.4 (rabbits) and Chapter 5]. Until recently, the mechanism for
SM ocular intoxication was thought to initiate from cytotoxic events fol-
lowed by tissue inflammation, however the authors suggest that evidence
now indicates that SM induced initiation of inflammation could precede
cytotoxicity.
SM is widely known for its vesicating properties. There are no documented
studies that conform to any standard regulatory test method for dermal
irritation/corrosivity. However, there is sufficient information from observa-
tions and tests in humans to establish SM as a primary irritant, and further
tests in animals are not required.
Table 2.4 Ocular
lesions in rabbits exposed to SM vapour. 105
Low dose (371 µg l−1 × 2 min High dose (419 µg l−1 × 2 min
Acute phase = 742 mg min m−3) = 838 mg min m−3)
First clinical signs Appear at +6 hours Appear at +3 hours
Tearing, lid erythema and oedema, conjunctival hyperaemia
and eye closure
4 hours Increased protein and decreased glutathione and ascorbic
acid levels in aqueous humour (note this is prior to onset of
clinical signs)
24 hours Lids swollen, eyes closed; conjunctiva and cornea oedematous
and corneal erosions observed
48 hours Epithelial denudation and marked stromal oedema of cornea;
cellular infiltration, mainly eosinophilic
48–72 hours Injuries most severe; extensive inflammatory response of eye-
lids, conjunctiva and cornea; deep corneal erosions
72 hours Spontaneous epithelial healing. Epithelial regeneration by
migration of non-injured corneal or conjunctival cells
1 week Majority of erosions healed; conjunctiva and eyelids still
oedematous and congested
Delayed phase 25% of eyes affected at 40% of eyes affected at the
the low dose high dose
Week 2 Recurrent corneal erosions, stromal sediments, corneal
neovascularisation
3 months As above, epithelium of clinically normal eyes was irregular,
exhibiting areas of hyperplasia, oedematous collagen
fibres
The chamber experiments carried out on human volunteers have defined

the lowest concentration at which very slight effects are observed on the eyes
and skin. These studies are reviewed in detail in Chapter 5 and summarised
briefly here.
2.2.4.2.1 Dermal. SM produces a dose dependent primary irritant

response, erythema, oedema and vesication/desquamation when it comes
into contact with the skin. The lesions produced by SM take many months to
heal and can produce long lasting depigmentation.
A number of studies in human volunteers have shown that the skin starts to
react to vapour in the dosage range 50–70 mg min m−3, and that almost all areas
of the body are vesicated (resulting in total tissue destruction and “burns” that
take many months to heal) above 600 mg min m−3. A no observed adverse effect
level (NOAEL) cannot be determined from any of these studies, since all expo-
sures, when those exposed were not wearing protective clothing, produced a
reaction in at least some subjects. Thus the Ct of 50 mg min m−3 (1.66 mg m−3 for
30 min) should be considered a lowest observed adverse effect level, but given
the authors comments that the effects of this exposure were difficult to discern
from other forms of mild irritation (e.g. abrasion of collars and cuffs) the level is
probably close to a NOAEL for irritancy of human skin by vapour.
48 Chapter 2
It is clear from these and other studies that hot, wet skin is more sensitive
to SM than warm dry skin, and there is some evidence to suggest that this
may be due to the presence of a layer of water (i.e. sweat) on the skin surface.
Studies carried out in the UK and India of the sensitivity of human skin to
contamination by liquid SM (applied in benzene solution that was assumed
to evaporate leaving liquid SM on the surface) established that across six dif-
ferent populations of young fit males, the mean effective dose, 50% (95%
CL) on human skin is 2.4 µg (2.2–2.5) with a probit slope of 2.4 (2.0–2.8). See
Chapter 5 Figure 5.6 for details.
2.2.4.2.2 Ocular Effects. There are two key studies in human volunteers
that quantify the effects of SM vapour on human eyes.106,107 Human eyes
respond in the same way as animal eyes with a dose dependent inflamma-
tory reaction that produces a conjunctivitis culminating in blepharospasm
at high doses. Although bulbar perforation is rare it does occur with high
doses of liquid or vapour. The lowest vapour concentrations to which the
eyes of human volunteers have been exposed are 0.06 mg m−3 for 24 hours in
one study and 6.25 mg m−3 for 2 min in another. These doses produced slight
bulbar injection.
2.2.5 Sensitisation
Sulzberger et al.108 reviewed experiments reported in the military literature
during WWII. The original reports of this work have not been located to
date. Studies in guinea pigs pretreated with dilutions of SM in ligroine (a
petroleum derivative) were described. Guinea pigs pretreated daily with eight
drops of 0.1% or 0.05% solutions of SM in ligroine ten times over 3 weeks
resulted in sensitisation when challenged 2 weeks later. Sensitised animals
reacted to a 0.1% droplet challenge when naive animals did not. The authors
reported that sensitisation could be induced by pretreatment with 0.1%,
0.05% and 0.02% solutions but not by 0.004% solution. Guinea pigs chal-
lenged with a single droplet of undiluted SM became hypersensitive but less
than those repeatedly challenged with 0.1% solutions. Holiday (reported by
Sulzberger et al.108) was also able to show “hypersensitivity” to SM in guinea
pigs pretreated by formol-killed tubercle bacilli (IP) followed 24 hours later
by 0.4 mg SM (IP). Holiday also induced sensitisation to the effects of SM by
scalding the skin sufficiently to produce oedema without ulceration, and by
repeated percutaneous application of SM in benzene to the same site prior
to challenge.
Moore (reported by Sulzberger et al.108) pretreated 46 guinea pigs daily
for 10 days with a drop of 0.1% solution of SM in benzene. All of the ani-
mals became sensitised, with 38 reacting to 0.003125% solution and 8 to
0.00625%. The same author demonstrated sensitisation by a burn produced
by a single drop of undiluted SM, but the animals were not sensitised to as
great an extent as when sensitised by repeated doses, reacting to an average
of a 0.038% solution.
Tests carried out in rabbits and rats did not produce sensitisation but the
methods used in these studies were not as rigorous as those in the guinea pig
tests described above.108
It is difficult to interpret these studies since the original reports have not
been located and appropriate control data are often absent from the review.
The criteria used to define “hypersensitive” and “sensitised” are also unclear.
It is also possible that “burning” the skin in itself may make skin generally
more sensitive to subsequent chemical insult.
2.2.5.2 Studies and Exposures in Humans

From the earliest studies it was apparent that people working with SM could
become sensitised to its effects. At the end of WWI Marshall109 reported that
after 6 months workers in his laboratory reacted to SM at concentrations in
the working environment that they had not noticed when they started work. A
series of studies were carried out in the UK and India during the 1930s defin-
ing how sensitive the normal and exposed population were to SM, and defining
the effects of temperature and ethnic origin.110–116 These studies are reviewed in
detail in Chapter 5 and were based on a test where 10 µl of SM diluted in dry ben-
zene was applied to the volar forearm using a specifically manufactured pipette.
These tests showed that men who had been injured by SM previously could
react to an amount of SM 1000 times lower than those who had not. This was
supported by a controlled study on volunteers reported by Moore and Rock-
man117 who showed that in addition to increasing the sensitivity of human
skin the degree of sensitisation was related to the number of times the sub-
ject had been burned.
These studies support the following conclusions:

1. SM is able to sensitise humans to its own effects on the skin;
2. There is a large variation in the normal sensitivity of the naive human
population to the effects of SM;
3. Indian skin is less sensitive than Caucasian skin to the effects of SM;
4. The sensitivity of human skin to liquid SM is dependent upon tempera-
ture and/or humidity in a similar way to that following exposure to SM
vapour;

2.2.6 Repeated Dose Toxicity

2.2.6.1.1 Oral. Sasser et al.118 dosed male and female Sprague–Dawley
rats (12 of each gender per dose group) with SM dissolved in sesame oil for
5 days per week for 13 weeks. The doses used were 0 (control), 0.003, 0.01,
50 Chapter 2
−1
0.03, 0.1 and 0.3 mg kg per day. No deaths occurred that were due to SM;
three animals died apparently from trauma caused by the dosing procedure.
Body weight gain was significantly depressed in both males and females
at the highest dose. Epithelial hyperplasia of the forestomach occurred in
5 out of 12 males in the high dose group and in 1 out of 12 that received
0.1 mg kg−1 per day, but not in any other treatment group. A small number
of squamous papillomas of the forestomach were observed in about 10% of
the intermediate and high dose groups; these lesions were considered to be
benign. No forestomach lesions were observed in any of the control animals.
No treatment related pathological lesions or changes in clinical chemistry or
haematology were reported. The authors suggested a NOAEL of 0.1 mg kg−1
per day since the forestomach epithelial hyperplasia observed in this group
was not significantly different from controls. However, in the two generation
reproductive toxicity study carried out by the same researchers119 treatment
by gavage for 17–22 weeks produced dose related forestomach lesions in
the 0.03, 0.1 and 0.4 mg kg−1 per day groups, suggesting that the effect may
become more pronounced with more prolonged exposure. On this basis,
0.03 mg kg−1 per day should be considered a LOAEL for forestomach lesions
in the rat after repeat exposure.
2.2.6.1.2 Inhalation. McNamara et al.80 studied 6 dogs, 12 rabbits, 30

guinea pigs, 140 rats and 140 A/J mice, housed in a chamber and continu-
ously exposed to 0.001 mg m−3 of SM vapour for between 1 and 52 weeks.
Temperature fluctuated with adjacent room temperature but the chamber
was heated during cold weather. The same species and numbers of animals
were housed in another chamber and exposed to 0.1 mg m−3 of SM vapour 5
days per week, 6.5 hours per day plus 0.0025 mg m−3 for the remaining 17.5
hours per day.
There were no signs of overt toxicity in animals exposed to 0.001 mg m−3
for up to 52 weeks. No details of how the vapour was generated or whether the
test atmosphere was monitored and analysed during the study were given. In
the 0.1 mg m−3 group ocular signs, corneal opacity, pannus, chronic keratitis,
vascularisation, pigmentation and granulation were observed after 16 weeks
of exposure and remained for the duration of the study.
Blood analysis was only carried out in dogs and rabbits, exposed to either
dose of SM. There were no effects on RBC count, total and differential WBC
counts, haematocrit or haemoglobin. There were also no changes in blood
clinical chemistry markers including: total protein, urea, serum glutamic
pyruvic transaminase, lactic dehydrogenase, alkaline phosphatase, creati-
nine, bilirubin, sodium chloride, potassium and carbon dioxide.
Rats, mice, guinea pigs, rabbits and dogs exposed to 0.001 or 0.1 mg m−3
SM for between 1 and 52 weeks were killed at pre-determined time points
and autopsied for pathology. Some of these species were used to assess car-
cinogenicity. Few lesions in this study could be directly attributed to SM.
“Sprague–Dawley–Wistar” rats and A/J mice were used to measure carcino-
genicity. In the rat study there was a significant increase in the number of
−3
animals developing tumours in the 0.1 mg m group (44%) compared with
low dose exposure (10%) and air controls (7%). In the mouse study there
was no significant increase in the number of animals developing tumours in
either group exposed to SM.
Guinea pigs exposed to air, 0.001 or 0.1 mg m−3 SM for between 1 and 52
weeks were used in sensitisation studies. At 1, 2, 4, 8, 16, 32 and 52 weeks
post challenge six animals per chamber were removed and challenged again
with 7.9 µg of SM in castor oil onto the skin. At this dose, sensitised animals
should show signs of erythema, oedema and necrosis. There was no evidence
of sensitisation.
The same animals were then challenged with 31.6 and 63.2 µg on different
spots; these doses produce erythema in normal animals. Erythema was seen
in the animals chronically exposed to SM suggesting that tolerance had not
occurred. There was also no evidence of sensitisation in beagle dogs when
respiration rates were measured after re-exposure to SM. There were no sensi-
tisation effects in rabbits after ocular exposure to 2 µg SM and then challenge
24 hours later with 2 µg, however a single dose of 20 µg did cause redness,
chemosis and corneal opacity without the need for a second challenge.
There are no controlled, repeat dose studies in humans, but the exposure
of workers to low doses of SM during its manufacture demonstrates that
repeated exposures can produce serious long term toxic effects.
Several studies suggest that workers who were chronically exposed to mus-
tard agents during their manufacture developed chronic, non-malignant
respiratory effects120–122 and that chronic respiratory disease may develop
in workers with only a few years’ employment.120 In all of these studies an
excess of influenza, pneumonia, bronchitis and asthma were reported even
amongst a population that had less than 3 years’ employment at the plant.
Similarly, workers exposed to SM and L in a Japanese production plant were
surveyed for respiratory morbidity (ill-health) 25 years after production had
ceased; the survey included investigation of chronic symptomatology and an
assessment of lung function. Highly exposed workers reported more chronic
bronchitis and had a lower respiratory capacity than both a less exposed
worker group and unexposed clerical workers.123,124 Morgenstern et al.125
described ten case studies of workers in US armaments factories in WWII
that clearly indicate the effects of occupational exposure to SM. After 6–12
months working in a shell filling factory, sensitive individuals presented
with symptoms of irritation of the conjunctiva and upper respiratory tract,
which progressed to photophobia, lacrimation, impaired vision and bleph-
arospasm. The senses of smell and taste were impaired or lost and there was
chest pain, retrosternal soreness, wheezing and dyspnoea. Symptoms also
included anorexia, vomiting, weight loss, general weakness, insomnia and
irritability. On removal from the source of exposure to SM, these symptoms
gradually subsided but the patients were left with a persistent paroxysmal
52 Chapter 2
hacking cough. The cough was precipitated by exposure to irritant dusts,
sudden changes of temperature or exertion, and produced small amounts of
mucoid or mucopurulent sputum. Over time, persistent bouts of bronchial
pneumonia resulted in production of definite clinical bronchiectasis.
None of the epidemiological studies of occupational exposure provide
estimates of exposure level. It has been reported that worker protection was
inadequate in Japanese factories and that employees were exposed to signifi-
cant levels of SM, and to lesser extent L.123 Conditions may have been better,
but still relatively poor, in British munitions factories.126
It is well documented that inhalation of SM causes injury of the respiratory
system. However, there is no direct evidence that addresses the issue of long
term respiratory effects in individuals who were exposed to very low levels
of SM and suffered no acute respiratory tract injury. Another study investi-
gated the long term respiratory effects in individuals claiming their symp-
toms were due to exposure to CWAs during the Iran–Iraq war.127 Out of 200
patients claiming respiratory problems only 77 veterans were entered into
the study some 15 years after exposure, based on their presence in the area
attacked at the time of an attack and not showing any signs or symptoms at
the time of exposure. A number of respiratory complaints were noted: 79%
had dyspnoea, 79% cough, 67% phlegm and 32% haemoptysis. All had nor-
mal chest X-rays while high resolution CT scanning showed air trapping and
bronchiectasis in some patients. Without good evidence of exposure and
full histories of what other chemicals the veterans admitted to the study had
been exposed to at the time, and in the intervening years, it is not possible
to establish a definite causal relationship between the symptoms described
and exposure to SM.
2.2.7 Mutagenicity
SM is a potent alkylating agent and reacts directly with DNA. It has been
shown to be mutagenic in in vitro studies in bacteria and also in mammalian
cells. Positive results were also obtained in vivo in rodents in both somatic
and germ cells. SM is clearly mutagenic in animals. SM was one of the first
chemicals investigated for its ability to cause direct chemical damage to DNA
and its genotoxicity has been extensively reviewed.6,24,45
2.2.7.1 DNA Damage In vitro

SM can react directly (without metabolic activation) with DNA due to its
potent alkylating properties. These were extensively investigated by Brooks
and Lawley during the 1960s and early 1970s.13,16,17,19 The majority of alkyla-
tions (about 75%) were monofunctional with the N7 nitrogen in guanine
resulting in the mono adduct. However, some cross-linking alkylation also
occurred and a range of minor products were produced. SM has been shown
to alkylate DNA from Escherichia coli,128 rat epidermal keratinocytes129,130 and
131
human WBCs, and to produce single strand breaks in DNA from bacterio-
phage lambda.132 SM has also been shown to produce DNA damage in bac-
teria using the “rec” assay,133 which uses a strain of Bacillus subtilis with the
recombinant genes recE4 and rec-45 turned out to maximise susceptibility
to mutagens.
2.2.7.2 Mutagenicity Studies In vitro

SM has been investigated for its ability to produce point mutations in bacte-
ria in the absence of an exogenous metabolic activation system. Salmonella
Typhimurium strains TA98 and TA100 were used as well as the original G46
strain. A clear positive result was obtained in G46 and also TA100.134
SM has also been shown to produce a high incidence of chromosomal
aberrations in a cultured rat lymphosarcoma cell line.135
2.2.7.3 Mutagenicity Studies in Drosophila

SM was the first chemical reported to induce mutations in the fruit fly Dro-
sophila melanogaster. Positive results were obtained in an assay that detects
mutations in the germ line of the insect in a sex linked recessive assay.136
2.2.7.4 In vivo Studies in Mammals

SM has been examined for its ability to induce micronuclei (an indirect indi-
cator of clastogenicity) in the bone marrow of mice following both oral and
IP administration.134 Single doses of SM were given by the IP route (4.8 and
10 mg kg−1) and one orally (10 mg kg−1). Bone marrow was harvested 24 hours
post dosing. A clear increase in micronuclei was seen following both oral
and IP dosing (in the latter case this was dose related), indicating that SM is
mutagenic.
SM has also been investigated for its ability to induce mutations in germ
cells in a dominant lethal assay.80 Adult male rats were exposed to SM vapour
at 0.001 and 0.1 mg m−3 for up to 52 weeks. Males were mated with unex-
posed females after 1, 2, 4, 8, 12, 24, 36 and 52 weeks of exposure. An increase
in dominant lethal effect was observed in the groups exposed to 0.1 mg m−3,
which was cumulative with time, reaching a maximum after 12 weeks of expo-
sure. The experimental detail given was limited and the authors reported
that the increase was not significantly different from unexposed controls,
although the control data were not presented in full.
An increase in chromosome aberrations and sister chromatid exchange was
reported in 1993 in former workers from a Japanese poison gas manufactur-
ing plant that was operational from 1927 to 1945. However, in view of the
54 Chapter 2
small numbers involved, the similarity of results between those involved in
SM production and other areas of the factory, and other limitations of the
study, no conclusions can be drawn regarding the mutagenicity of SM in
humans.137
2.2.8 Carcinogenicity
There are a limited number of carcinogenicity studies of SM in animals. The
International Agency for Research on Cancer (IARC) has classified SM as a
human carcinogen.138
2.2.8.1.1 Oral. Sasser et al.119 dosed male and female Sprague–Dawley rats
as described in Section 2.6.1.1 at doses of 0 (control), 0.003, 0.01, 0.03, 0.1
and 0.3 mg kg−1 per day. No deaths occurred that were due to SM, but epithe-
lial hyperplasia of the forestomach occurred in 5 out of 12 males in the high
dose group and in 1 out of 12 that received 0.1 mg kg−1 per day, but not in
any other treatment group. A small number of squamous papillomas of the
forestomach were observed in about 10% of the intermediate and high dose
groups; these lesions were considered to be benign. No forestomach lesions
were observed in any of the control animals. In a two generation reproduc-
tive toxicity study carried out by the same researchers119 treatment by gavage
for 17–22 weeks produced dose related forestomach lesions in the 0.03, 0.1
and 0.4 mg kg−1 per day groups, suggesting that the effect may become more
pronounced with more prolonged exposure. These findings support the view
that SM is a site of contact carcinogen, particularly as this was a 13 week
study and a longer duration study would have been expected to result in a
higher tumour yield.
2.2.8.1.2 Inhalation. Heston139 showed that exposure to SM vapour

increased the incidence of pulmonary tumours in mice from an incidence
of 27% in controls to 49% after an exposure to a dosage that produced skin
damage at the time of exposure and weight loss for 10 days after exposure.
The dosage of SM to which these animals were exposed was not quantifiable
from the method of exposure used.
In a large study of the effects of long term exposure to low levels of SM,
McNamara et al.80 exposed a number of species to 0.001 mg m−3 continuously
for 52 weeks and to 0.1 mg m−3 for 6.5 hours a day for the same period (and
0.0025 mg m−3 for the remaining hours of the day). No details of how the
vapour was generated or whether the test atmosphere was monitored and
analysed during the study are given. The results indicated that exposure to
0.001 mg m−3 for 52 weeks produced squamous and basal cell carcinomas in
5 out of 48 animals (10%) while exposure to 0.1 mg m−3 for 8 hours per day for
52 weeks produced tumours in 25 out of 57 animals (44%). No tumours were
recorded in the respiratory tract. This, compared with the tumour rate in the
controls of 2 out of 27 animals (7%), clearly indicates that SM is tumorigenic
at the higher dose over an exposure period of 52 weeks.
The main weakness of this study is that its duration, 12 months, is not the
lifetime of rodents and thus the results are likely to have underestimated
the true carcinogenic potential of SM. Since carcinogenicity tests in animals
should be able to detect tumours appearing near the end of the lifespan
(2 years for laboratory rodents), this study does not satisfy modern stan-
dards.140 Also, although the study used large numbers of animals, because
of the interim sacrifices, relatively few animals were remaining at 12 months,
which further reduced the power of the study to detect a carcinogenic effect.
2.2.8.1.3 Other Routes of Exposure. Heston141 described studies of the

incidence of pulmonary tumours in mice treated intravenously with nitrogen
and mustards. The author highlights difficulties in the precise estimation of
the dose injected because of the rapid hydrolysis of the agent in the chosen sol-
vent (water). Mice (equal numbers of males and females) were given four injec-
tions on alternate days of 0.25 ml of a 0.06–0.07% solution of SM in distilled
water. Animals were killed 16 weeks after the first injection and the number
of nodules on the lungs counted. In the first experiment nine males and four
females died soon after injection whereas in the second the dosing solution
was prepared with the precise amount of SM, as opposed to a slight excess,
and only one male animal died before sacrifice. In both experiments there was
a drop in body weight compared with controls. The results of the second exper-
iment are the more reliable and nodules in the lung were observed in 68.1%
of the animals in the test group compared with 13% in the control group (an
average of 1.09 nodules in the test group compared with 0.13 in controls). It
should be noted that the author cautions the reader about not being able to
precisely estimate the dose injected, and that SM has a very short half life in
aqueous solution so the precise dose given is not known.
In a subsequent study, Heston142 injected several groups of mice, of mul-
tiple strains, subcutaneously with SM in olive oil (5 or 6 weekly injections of
0.05 ml of 0.05% solution). One experiment with 0.1 ml of 0.1% solution was
discontinued after one injection due to the poor condition of the animals.
Not all of these experimental groups had simultaneous control groups. The
injections produced a low incidence of sarcomas at the site of injection; one
mammary tumour and one rhabdomyosarcoma. The tumours at other sites
were primarily mammary and pulmonary tumours with some hepatomas
and three cases of lymphocytic leukaemia that the authors identify as hav-
ing a low spontaneous rate in these strains of mice (strains C3H and C3Hf
derived from A/J mice were used). The incidence of tumours in this study
is difficult to interpret due to the poor study design. The total incidence of
tumours, local and systemic, irrespective of sex and strain is 44 out of 46 con-
trols and 73 out of 121 SM treated mice, giving a total incidence of 96% in the
control animals and 60% in those treated with SM. It is not possible to assign
any real significance to the results of this study. The author also reported that
the nodules could be transplanted to naive mice and continued to grow.
56 Chapter 2
There is epidemiological evidence for the carcinogenic effects of SM from
soldiers exposed during combat in WWI and in the Iran–Iraq war, volunteers
in WWII trials and workers exposed during the manufacture of SM.
In cases of occupational and/or battlefield exposure to SM, the individuals
affected will potentially have been exposed by multiple routes, although pre-
sumably the inhalation and dermal routes would predominate.
Early studies of the epidemiology of neoplastic disease in UK veterans of
WWI showed an increase in the incidence of cancers of the respiratory tract,
but no other organ.143 This increase was not significant at the time the study
was conducted (1952). A retrospective cohort study comparing 2718 WWI US
veterans exposed to SM with a similar control group and 1855 men who suf-
fered pneumonia during the influenza pandemic of 1918–1919 showed that
there was a slight increase in deaths due to lung cancer in those exposured
to SM.144 Further analysis showed that this small increase in the incidence
of death due to lung cancer (2.5% compared with 1.9% in the control group)
supports the case for a carcinogenic effect, when the influence of smoking
was excluded.145 Epidemiological studies of the US veterans from WWII who
were exposed in chamber tests146 concluded that exposure to levels of SM
“sufficient to cause skin reactions (erythema, vesicles and ulceration) was
not associated with an increase in any cause specific mortality”.147 It should
be noted that the volunteers in the WWII chamber trials wore respiratory
protection during exposure, so any exposure of the respiratory tract to SM
would have been accidental and probably very low.
In a study of 40 veterans of the Iran–Iraq war who showed signs of poison-
ing at the time of exposure, 16–20 years after exposure, Hefazi et al.91 failed
to show any increase in respiratory tract carcinomas in spite of recurrent
respiratory symptoms. Given the small increase in incidence shown above, a
group size of 40 is unlikely to be large enough to show an effect.
In contrast to these observations, retrospective studies of workers from sev-
eral different manufacturing sites for SM indicate an increase in the incidence
of respiratory tract carcinoma. Workers from the OkunoJima poison gas fac-
tory have been extensively studied. This factory manufactured SM, L, diphenyl-
cyanoarsine, hydrocyanic acid, chloracetophenone and phosgene between
1927 and 1945, before being dismantled between 1946 and 1947.124 Most of
the workers in the plant suffered respiratory irritation during their time at
the plant, which developed into chronic bronchitis the same as that already
described herein. Workers reporting to the Hiroshima School of Medicine
between 1952 and 1981 suffered from cancers of the tongue, sinuses, pharynx,
larynx, trachea and bronchus. Cancers of the oesophagus, liver, intestine and
urinary bladder were also reported.148–151 Histologically these tumours were
squamous cell carcinomas, undifferentiated tumours or adenocarcinomas.
The early epidemiological studies of these workers showed some evidence of
an increase in respiratory tract carcinomas.123 Later studies of the same popu-
lations between 1952 and 1987 indicated that exposure to war gases, including
SM, added significantly to the increasing incidence of respiratory tract carci-
nomas with age.149 A major confounding factor in drawing conclusions from
these data is exposure to multiple gases, although it is unlikely that hydrocy-
anic acid, phosgene, chloracetophenone or diphenylcyanoarsine are respon-
sible for the increased incidence of cancer, since they have not been shown to
be carcinogenic in any other study. It is not, however, possible to conclude that
either SM or L individually is the cancer inducing agent.
Studies of the causes of death of workers in the UK who manufactured
SM during WWII revealed a significant increase in the incidence of cancer
of the larynx, but not of any other organ.121 This conclusion was based on
two deaths from cancer of the larynx and one from cancer of the trachea
(against an expected incidence of 0.4) in a group of 511 male and female
workers. A study of a much larger group, 3354 workers, showed large and sig-
nificant increases in cancer of the larynx (11 deaths observed, 4.04 expected),
pharynx (15 deaths, 2.73 expected), all other buccal cavity and upper respi-
ratory tract sites (12 deaths, 4.29 expected), and lung cancer (200 deaths,
138.39 expected).120 The same study found that the incidence of cancer of the
pharynx and lung was significantly related to length of employment. Signif-
icant increases in oesophageal and stomach cancer were also reported. This
appears to be a reliable study and the results point very strongly to a carcino-
genic potential for SM.
2.2.8.3 Conclusions for Carcinogenicity

To date the animal studies conducted to address the carcinogenicity poten-
tial of SM have been poorly designed. There has only been one long term
inhalation exposure study but this did not describe the exposure method-
ology in sufficient detail to confirm exposure concentrations. The results
clearly indicate that daily exposures for 6.5 hours over 12 months to 0.1 mg
m−3 SM led to a high incidence of malignant skin tumours in rats, with skin
tumours being apparent as early as 12 weeks into the study. This is indica-
tive of a potent carcinogen. A 13 week oral dosing study in rats also showed
evidence for carcinogenic potential based on the development of hyperplasia
and squamous papillomas of the forestomach. A 13 week oral dosing study
in rats also showed evidence for carcinogenic potential based on the develop-
ment of hyperplasia and squamous papillomas of the forestomach. A major
short coming in assessing the carcinogenic potential of SM is the lack of ani-
mal studies of appropriate duration (18–24 months in rodents).
Epidemiological studies in veterans of conflicts in which SM has been
used are all limited by the small size of the groups investigated (in the con-
text of detecting a small increase in cancer incidence), which results in all of
the studies failing to show statistically significant differences. A large num-
ber of military volunteers were exposed to SM between 1918 and the early
1970s (see Chapter 5 for a more detailed discussion), and to date no follow
up study of any of these groups has shown a significant increase in any can-
cer, or indeed any cause of mortality.
58 Chapter 2
The only statistically significant evidence of increased cancer is in workers
who were exposed during the manufacture of the agent (i.e. long term expo-
sure to concentrations that may have caused effects at the time of exposure).
There are, however, no reliable data on how these workers were exposed, via
what route, at what concentration and over what period of time. Although
several studies of manufacturing workers have been carried out at various
times after exposure ceased, a statistically significant increase in the inci-
dence of pulmonary tumours alone was only shown 40 years after exposure.
On balance, there is sufficient evidence from animal studies and human epi-
demiology to show that SM is carcinogenic, producing tumours in tissues at
the site of contact. No clear dose–response relationship for carcinogenicity
can be constructed from the data available. There are no reliable studies of
the potential for SM to cause tumours following a single exposure.
2.2.9 Toxicity for Reproduction

A number of studies have been conducted to investigate the effects of SM on
human and animal reproduction. To date none of these studies have pro-
duced evidence that SM expresses any significant reproductive toxicity at
doses that fail to cause significant acute toxicity in either parent. However, to
date no study has been conducted on the incidence of abnormalities in the
offspring of veterans from previous conflicts in which SM was used or muni-
tions factory workers.
2.2.9.1.1 Developmental Toxicity. Rommereim and Hackett152 adminis-
tered oral doses of SM at 0–2 mg kg−1 on gestation days 6–15 inclusive (rat)
or 0–0.8 mg kg−1 on gestation days 6–19 inclusive (rabbit). Maternal toxicity
was observed at all dose levels in the rat and foetal toxicity (reduced ossifica-
tion and skeletal abnormalities) was observed at the highest dose level only.
Maternal death, reduced body weight and decreased haematocrit were seen
at the highest dose in the rabbit; this was associated with decreased birth
weight, but no other abnormalities were observed in the offspring. The study
concluded that SM was not teratogenic in rats or rabbits since foetal abnor-
mality only occurred at dose levels that induced maternal toxicity.
2.2.9.1.2 Fertility. In a 42 week two generation study in the rat,119 groups

of 27 females and 20 males/group/generation were treated by gavage at 0,
0.03, 0.1 and 0.4 mg kg−1 SM for 13 weeks prior to mating and throughout
gestation, parturition and lactation. Males were sacrificed at the birth of the
offspring. At weaning selected F1 generation animals continued in the study
and were treated in the same way as the F0 generation, and males were sacri-
ficed at the birth of the offspring. There were no adverse effects on reproduc-
tive performance or fertility in either male or female rats except for an altered
−1
gender ratio in the highest dose group (0.4 mg kg per day). However, growth
of the offspring was depressed during lactation. Dose related lesions of the
epithelium of the forestomach were seen (acanthosis and hyperplasia) at all
dose levels in both F0 and F1 generations, although effects were mild at the
lowest dose of 0.03 mg kg−1 per day. Benign neoplasms were found in 8 out of
94 animals at 0.1 mg kg−1 per day and in 10 out of 94 animals at 0.4 mg kg−1
per day. Based on the incidence of forestomach lesions seen at all doses, 0.03
mg kg−1 per day can be identified as a LOAEL for parental toxicity.
Ghanei et al.153 investigated the effects of SM on human fertility in a retro-
spective cohort study that examined civilians (115 couples) exposed to liquid
SM by aerial bombardment of the city of Sardasht in 1987, during the Iran–
Iraq war. The definition of infertility in this study was “the failure to con-
ceive after 12 months of unprotected intercourse after marriage”. Individuals
included in the study had shown symptoms of acute SM exposure following
the attack. Patients with a history of chronic diseases that may cause loss
of sexual function (e.g. diabetes, hypertension, heart disease, vascular prob-
lems, multiple sclerosis and spinal cord injuries) were excluded. In 88 cases,
only one partner had been exposed to SM, whereas in 27 cases both partners
had been exposed. The fertility of these couples was compared to the world-
wide fertility index. The results of the study showed that exposure to SM at
the time at which couples were attempting to conceive did not correlate with
decreased fertility. However, the results should be interpreted with some
caution since the experimental design limited the definition of infertility to
a single 12 month period and no control group of unexposed couples was
included.
A study by Saferinejad154 examined the testicular effect of SM on 81 Ira-
nian men (28–41 years old) who had been exposed to SM and had the pre-
senting symptom of infertility. Forty-four patients were described as being
severely injured, 20 moderately and 17 mildly, but no quantitative exposure
data were available. Semen analyses, serum hormonal determinations (lutei-
nising hormone, follicle-stimulating hormone and testosterone) and genital
examinations were completed for all patients, as were testicular biopsies in
24 patients. Azoospermia and severe oligospermia were diagnosed in 42.5%
and 57.5% of patients, respectively, mostly from the severely injured group.
Hormone studies revealed an elevated plasma follicle-stimulating hormone
level and normal plasma luteinising hormone and testosterone concentra-
tions. Testicular biopsy showed selective atrophy of the germinal epithe-
lium, intact Sertoli cells, and normal-appearing Leydig cells. Subjects mildly
exposed reported restored fertility within 3 months of exposure, whereas
those severely injured exhibited long term infertility. The authors conclude
that mustard gas can cause defective spermatogenesis years after expo-
sure. However, while this study is useful in describing general trends in SM
60 Chapter 2
induced infertility, it has little epidemiological value due to selection based
on infertility.
Azizi et al.155 described the effects on hormones associated with repro-
ductive function in Iranian veterans following battlefield SM exposure. All
hormone levels returned to normal by 12 weeks after exposure, but a low
sperm count (<3 × 106 ml−1) remained in 66% of those studied 1–3 years after
exposure.
A possible explanation for the conflicting results found in the above stud-
ies could be the level of exposure to SM or simultaneous exposure to an
unidentified factor. It is also possible that using infertility as a selection cri-
teria for admission into the first study reviewed153 failed to take into account
those individuals whose fertility had not been affected by exposure. It may
be that only relatively high doses of SM are directly gonadotoxic in humans,
although it must be emphasised that there are no experimental data avail-
able to support this hypothesis.
2.2.10 Summary of SM Toxicology

SM damages all of the surfaces of the body that it comes into contact with.
It produces inflammation of the eyes, skin, gastrointestinal and respiratory
tracts in a dose dependent manner, which culminates in tissue destruction
and necrosis. Macroscopically the toxicity of SM manifests as the formation
of an injury pathologically similar to a thermal burn, but is slower to develop
and heal. The skin shows all of the signs of primary irritation and blisters at
higher doses in humans. The respiratory tract produces a necrotic slough
that coats the inside of airways and can slough off to block lower airways. In
the deep lung, damage can result in the breakdown of the lung lining and
pulmonary oedema. Similarly in the gastrointestinal tract the lining cells
become necrotic and slough off into the lumen producing diarrhoea and
vomiting. The eyes are irritated to produce conjunctivitis, photophobia and
blepharospasm. Corneal opacity can result, with keratitis either immediately
after exposure or after a delay of some years.
SM is a primary irritant in man and may sensitise humans to subsequent
applications.
SM is a genotoxic carcinogen and this must be considered a critical health
effect in health based risk assessments. Although SM is a mutagen in ani-
mals, it does not produce toxicity to reproduction in animal tests at doses
that are non-toxic to the male or female parent.
2.3 Lewisite
The development of chemical weapons during WWI led to an attempt to com-
bine the lethal effects of agents like phosgene with the longer term incapaci-
tating effects of SM. The result was the weaponisation in 1918 of a chemical,
first synthesised in the 1800s, named Lewisite after Capt. Winford Lee Lewis
Figure 2.4 The

structures of LI, II, III and L oxide.
who developed it. L is an arsenical agent156–158 that was produced in quantity

too late to be used in WWI; it was on its way to Europe when the armistice
was declared. However, it was subsequently manufactured in quantity by a
number of nations including the USSR, Japan and Germany. There is not as
much toxicological information in the published literature for L as there is
for SM. What there is indicates that L has similar actions to SM but it exerts
its actions more rapidly, causes an immediate sensory irritation and the tis-
sue damage produced heals more quickly. L is chemically more complex than
SM, existing as cis and trans isomers about the double bond, and as L I, II and
III (Figure 2.4) depending on the number of 2-chlorovinyl groups attached
to the arsenic atom (see Goldman and Dacre159 for a review of L chemistry).
This section reviews the toxicology of L I, although some of the early studies
quoted may have investigated a mixture of L I, II and III plus L oxide because
the analytical confirmation of chemical identity was difficult.
2.3.1 Toxicokinetics
There are very limited quantitative toxicokinetic data on L. Although there
are no published estimates of the diffusion rates of L through the skin,
observations from acute toxicity studies indicate that it penetrates rapidly.
Studies of the kinetics of elimination of arsenic after subcutaneous injection
of L in rabbits show that it is eliminated with a half life of 55–75 hours.160
The primary metabolite of L is 2-chlorovinyl-arsenous acid, which is formed
by hydrolysis. This metabolite, like the parent compound, is able to bind
to sulphydryl containing proteins. It is this property that is believed to be
responsible for the toxicological effects of L, although no primary biochemi-
cal lesion has been identified. In rabbits the apparent volume of distribution
62 Chapter 2
of L is several litres per kilogram indicating extensive tissue distribution or
binding. This is consistent with binding to proteins and/or other tissue con-
stituents being a major route of elimination from the blood, as is indicated
by its binding to haemoglobin in the blood.161
2.3.2 Acute Toxicity

The drive to define the toxicity of CWAs during WWII led to an extensive
programme of research in the UK and USA. From early studies115,162 it was
clear that the more rapid injury produced by L and the immediate sensory
irritation removed the latency of response characteristic of SM injury. There
is also systemic toxicity associated with exposure to L that is similar to that
produced by arsenic. Some authors163,164 conclude that the arsenic content of
L is responsible for its systemic toxicity, but others165 point out differences
that indicate alternative mechanisms of toxicity are involved. It is tempting
to speculate that the chlorovinyl group produces a different tissue distribu-
tion for L compared with arsenic and this may be responsible for the dif-
ferent toxicity profile, but no specific studies of the toxicokinetics of L have
been carried out.
The acute toxicity of L was described in open publications and reviews
during the 1940s and some of the defence reports that give more detailed
accounts are now in the public records. L has similar effects to SM when
applied to the skin or administered by injection.163,164 It is acutely lethal at
doses of 5–25 mg kg−1 percutaneously, 1–2 mg kg−1 subcutaneously and 0.5
mg kg−1 intravenously (Table 2.5) in a range of species. By inhalation, the
LCt50 is 120–2800 mg min m−3 for a 10 min exposure in a range of species
(whole body exposure). Experiments in mice show that L has approximately
the same lethality by inhalation alone as by body only exposure. Due to the
immediate irritation produced by L the acute toxicity by inhalation should be
treated with caution because animals modify their breathing in the presence
of irritants. The results of inhalation of L vapour are an immediate irritation
followed by acute inflammation of the upper respiratory tract and later pneu-
monitis and pulmonary oedema within the first 24 hours that is followed by
bronchopneumonia.164,166 The lesions are very similar to those described for
SM inhalation.
The species specified in Table 2.5 also show considerable damage to the
nasal epithelia; inflammation and sloughing of the lining of the nose will
cause difficulties in breathing for obligate nasal breathers that are not seen
in humans. It should be noted that the main inhalation study quoted by
Gates et al.156 (i.e. Cameron et al.166) may be flawed in that the dosages were
achieved by varying exposure time to two different concentrations. To use
these data it is necessary to assume that the toxic load exponent is 1, i.e.
Haber’s law applies; however, there are not sufficient data to support this
Table 2.5 LD
50 and LCt50 of L by various routes of administration in several species.
Vapour exposure (mg min m−3)
Subcutaneous Percutaneous (mg
Species Intravenous (mg kg−1) (mg kg−1) kg−1) Total Inhalation only Body only
164
Goat 15 125
24 156
10 156
Dog 2 164 15 164 140 3000
38 156 3000
70 156 4000
Rabbit 0.5 164 2 164 6 164 120 1500
1.8 165 5 156 150
6 156
6 156
Guinea pig 1 164 15 164 1000 2000–2500
12 156 470
Rat 1 164 24 164 1500 2000
24 156 580
15 156
24 156
20 156
Mouse 15 156 900–1400 1400–1500 1200–1900
2800 1600 300
1500 1500 7000
2500–2800
500
Compiled from the sources indicated or, for the vapour exposure values, from the various sources examined and reported by Gates et al.156
63
64 Chapter 2
assumption. Indeed, the difficulties caused by the immediate irritant effect
of L suggests that the toxic load exponent would be higher than 1.
The pathology of L poisoning in laboratory animals (mice, rats, guinea
pigs and rabbits) has been described in some detail after intravenous and
percutaneous exposure.163,164 There was a marked variation in the patho-
logical changes induced by L between species. For instance, congested and
occasionally haemorrhagic pulmonary lesions were not common in guinea
pigs and mice but were often observed in rats and rabbits. These pulmonary
lesions appeared rapidly after intravenous injection, but not after percutane-
ous application. Small areas of alveolitis and bronchiolitis were seen for sev-
eral days after dosing and both emphysema and oedema were observed. In
the guinea pig and the rabbit, but not the rat and the mouse, hepatic and bili-
ary pathology developed between 3 and 12 hours after percutaneous applica-
tion of L. The liver became very congested and the gall bladder and bile duct
became distended with watery bile. This progressed to areas of necrosis of
the mucosa of the gall bladder and liver such that the authors described the
liver as having areas of coalescence forming infarct like areas. Once initiated
the hepatic necrosis progressed rapidly in animals that succumbed to poi-
soning with the liver condition returning to normal within 4 days in surviv-
ing animals. Liver damage was also observed in guinea pigs and rabbits after
intra-peritoneal, subcutaneous or intravenous injection.
In summary, the major systemic pathological changes produced by L are to
the liver and biliary system, with some transient renal changes. Any changes
to the cardiovascular and nervous system were limited to congestion or as
secondary effects to other pathologies (e.g. dilatation of the right side of the
heart in animals dying within 24 hours probably resulting from damage to
the lungs).
A condition named “Lewisite Shock” has also been described167 that is
associated with loss of blood volume and an associated increase in RBC
count and haemoglobin concentration. This is accompanied by a decrease
in plasma volume and plasma proteins, suggesting that movement of pro-
tein through damaged membranes may alter the hydrostatic pressure in the
tissues and cause fluid to move into the tissues. The movement of fluid is
consistent with the observation of “copious jelly-like oedema” at the site of
application. The condition developed within 24 hours of dermal application
of L to animals and persisted for up to 4 days. The authors point out the
marked similarity between this condition and traumatic shock. Certainly,
the effects of fluid loss from the circulation by blood loss and the pathology
described above would be expected to be very similar, if not identical.
Cameron et al.164 described the pathology of skin injury in humans after the
application of liquid L. In biopsies from volunteers who had liquid L applied
to their skin, the epidermis separated at the dermo-epidermal junction and
no vestiges of the dermal tissue adhered to the underside of the blister roof.
Epidermal cell structure on the roof of the blister was predominantly lack-
ing, some hair follicles and sweat ducts crossed the blister and were substan-
tially damaged. In the deeper dermis there was a band of inflammatory cells
beneath the blister that were more numerous towards the edge. The collagen
bundles showed clear separation, with fibrin deposition in the dermis and in
the blister fluid. Capillary congestion was very marked throughout the der-
mis with coagulation necrosis in the centre of the lesions. These results were
very similar to those seen in experimental lesions produced on rabbit ears by
the same authors.163,164
Daily et al.168 described a comparison of L lesions with SM and nitro-
gen mustard (HN3) injury on the skin of human forearms. In 12 volunteers
lesions were raised using a vapour cup applied to the skin for varying times
(HN3 100 min, SM 10 min and L 6 min) to raise lesions of comparable sever-
ity for each of the three agents. Small amounts of liquid (0.5 mg and 0.5 µl)
were also applied to the skin using a “platinum pointed drod” (a purpose
built applicator designed to deliver a specific amount of agent, the design
of which was not described). The lesions produced were tense peduncu-
lated vesicles with a small area of erythema around the base that started to
subside after a few days and then showed a palpable jelly-like mass within
the blister. Smears of the blister fluid showed numerous polymorphonucle-
ocytes, but no bacteria. The SM and L lesions peaked in severity at about
the same time (39 and 42 hours, respectively) but the SM lesions took lon-
ger to heal. L also produced a secondary reaction about 14 days after appli-
cation, consisting of a marked erythema, numerous small vesicles and
vesicles in the area around the primary burn site. SM did not produce a
secondary reaction. The authors concluded that the L lesions were not as
severe as those produced by SM in the same study, but a full dose response
relationship was not determined precluding any conclusions about the true
comparative potency.
2.3.3 Ocular Toxicity

The effects that L has on the eyes are similar to those of SM with the addi-
tion of immediate irritation. This immediate effect results in a reflex bleph-
arospasm that limits exposure and caused early reviewers to conclude
that L had less effect than SM.159 The pathology of L induced ocular injury
has been described by a number of early reviews of the defence litera-
ture,156,159,169–171 but much of the original source data are unobtainable. The
reviews however describe a destructive ocular lesion from small amounts of
liquid L instilled into rabbit eyes. A 0.1 mg drop caused perforation of the
cornea in 75% of animals treated and corneal haze in the remainder. Gates
et al. report US defence sources as determining that doses of 0.01–0.02 mg
per eye of liquid L will cause permanent damage to rabbit eyes equivalent to
that caused by 0.1–0.2 mg of SM. The same sources report that a nominal Ct
of 2800 mg min m−3 of L is required to produce moderate corneal damage in
dogs and a Ct of 5500 mg min m−3 is required for a destructive lesion. These
66 Chapter 2
Cts are high compared with those of SM that cause effects in humans (see
Chapter 5).
In rabbit eyes histopathological changes are reported within 10–15 min
after instillation.171 There was swelling of the corneal basal epithelial cells
and separation of the epithelium from the underlying stroma. Over the
next hour the corneal epithelium was progressively lost and vacuolation
was observed around stromal nuclei as oedema fluid accumulated, produc-
ing separation of stromal fibres. Blood vessels in the conjunctiva, iris and
ciliary bodies became congested. By 6 hours the corneal epithelium had
completely detached and the conjunctiva was oedematous with polymor-
phonuclear leukocyte infiltration, stromal nuclei numbers were reduced,
and the iris and ciliary bodies were oedematous. By 24 hours the only area
of the stroma showing any nuclei was the limbus area where there was a
polymorphonuclear cell infiltration. The fibres of the stroma were less dis-
tinct. These changes became increasingly severe over the next 7–10 days as
the stroma became increasingly necrotic and ulcerated. There was vascu-
larisation in the deep stroma and at higher doses the bulb perforated. The
whole anterior segment of the eye was still severely inflamed but in less
severely injured eyes epithelial regeneration began with the conjunctival
oedema reducing. By 14 days small perforations had closed, the cornea was
covered by an irregular epithelium, conjunctival oedema had subsided and
the stoma was showing signs of healing with numerous fibroblasts, round
cells and small to medium sized blood vessels in the middle layer of the
stroma. The eye damage caused by L appeared more immediate and severe
than that caused by SM, but there were fewer recurrences of symptoms at
later times.
No studies of the effect of L on human eyes, or reference to them, have
been located.
2.3.4 Repeated Dose Toxicity

Sasser et al.172 reported a 90 day repeated dose toxicity test. Sixty animals of
each sex (10 per dose level per sex) were treated with 0.01, 0.1, 0.5, 1.0 or 2.0
mg kg−1 L for 5 days per week for 13 weeks in a constant volume of 1.67 ml kg−1.
There was no change in body weight gain, although at the highest dose, serum
protein, creatinine, serum glutamic oxaloacetic transaminase and serum glu-
tamic-pyruvic transaminase were significantly decreased in the males; and
lymphocytes and platelets were significantly increased in the females. The
result of most biological significance was a treatment related lesion in the
forestomach at 2.0 mg kg−1 with necrotic stratified epithelium infiltrated by
neutrophils, macrophages and fibroblasts. These lesions showed proliferation
of neo-capillaries, haemorrhage and oedema; and the mild acute inflamma-
tion seen in the forestomach supported the conclusion that the pathological
changes were due to prolonged local irritation of the forestomach produced by
L when gavaged. The authors concluded that the NOAEL was between 0.5 and
1.0 mg kg−1 by oral administration.
2.3.5 Mutagenicity
Jostes et al.173 showed L to be cytotoxic and clastogenic in cultured Chinese
hamster ovary (CHO) cells exposed to L for 1 hour, washed and then main-
tained for 24 hours. After 24 hours, the cell survival was characterised by a
D37 (dose at which 37% of the cells survive) of 0.5 µM (extrapolation num-
ber 2.5), but the mutagenic response of the hypoxanthine-guanine phos-
phoribosyltransferase (HGPRT) locus was not significantly increased over
the 0.12–2.0 µM range. Sister chromatid exchange was weakly positive over
the 0.25–1.0 µM range but not significantly different from controls. Chro-
mosomal aberrations were significantly induced at 0.5, 0.75 and 1.0 µM. The
authors were reluctant to conclude that L was not a mutagen since their tests
did not exclude the possibility that L was mutagenic at multiple loci, but it
was not mutagenic in CHO cells at the HGPRT locus.
2.3.6 Toxicity for Reproduction

Bucci et al.174 tested the effects of L on male reproductive capacity in a dom-
inant lethal mutation test. Male CD rats were given 1.5, 0.75, 0.375 mg kg−1
L or sesame oil (controls) by oral gavage (20 per dose group) for 5 days. Each
male was then mated with two virgin females per week for 10 weeks to cap-
ture the effects on spermatogenesis at various stages. Females were killed at
14 days after mating and the number of corpus lutea was determined. The
uteri and their contents were also examined. Males were killed at week 13,
sperm motility measured and the histopathology of the testis evaluated.
No effects were observed on male or female reproductive indices, except in
the positive controls (100 mg kg−1 ethyl methanesulphonate IP on day 5).
The authors identified a NOAEL of 1.5 mg kg−1 for a dominant lethal effect,
although this was the highest dose group and no effects were observed so it
is probably an underestimate of the true NOAEL.
In a 42 week two generation reproductive toxicity test Sasser et al.175 treated
three groups of male and female rats prior to, during and after mating until
the birth of the offspring with L (0.1, 0.25 and 0.6 mg kg−1 per day by oral
gavage). The dams were treated with L during lactation. Offspring were ran-
domly selected at weaning to continue the study and were given L continu-
ously through to gestation. Sesame oil was given to separate control animals.
No adverse effects on reproductive performance, fertility or reproductive organ
weight were found at any dose in either generation. There were no adverse
effects on offspring. The overall growth of F0 and F1 females was significantly
reduced in the 0.25 mg kg−1 dose group. This effect was also produced to a
lesser degree in the 0.6 mg kg−1 dose group but the low number of survivors in
this group compromises any conclusions drawn. Deaths in this group were not
treatment related. The authors attributed the early deaths in all dose groups
(15 out of 45 at 0.6 mg kg−1 and 4 out of 45 at 0.25 mg kg−1) to respiratory
tract damage caused by aspiration of L during dosing or by gastro-oesophageal
reflux. The NOAEL for reproduction was higher than 0.6 mg kg−1 in this study.
68 Chapter 2
2.3.7 Summary of L Toxicology

There is less known about the toxicology of L than of SM. However, what is
known indicates that L is a potent vesicant capable of causing direct damage
to any tissue it comes into contact with, in vapour or liquid form. In this
respect it is identical to SM, but it produces more tense blisters with jelly-like
blister fluid on the skin and inhalation of the vapour causes an immedi-
ate irritant response followed by inflammation of the respiratory tract and
pulmonary damage that can be life threatening. L differs from SM in that it
produces an immediate dermal irritancy response and the lesions produced
appear to be more severe and heal faster. L also produces a larger disturbance
of fluid balance than SM resulting in a “shock” like condition characterised
by loss of blood volume and haemoconcentration. L does not appear to be a
mutagen and does not cause reproductive toxicity at doses that cause weight
loss in the dams.
2.4 Conclusions
Although banned by international agreement and controlled by the CWC, ves-
icant agents remain available to countries and non-state actors and terrorist
groups that wish to use them, because they are relatively easy to make and the
methods to make them have been published. As part of our defence against
them, a knowledge and understanding of their toxicology is essential. The
most commonly used vesicant, SM, is a potent vesicant that is also a genotoxic
carcinogen and mutagen. SM is not toxic to the reproductive system at doses
that do not cause effects in the parents and can cause central nervous system
effects at high doses. The main toxic effects of acute and repeated dose expo-
sure, however, are related to the massive inflammatory reaction it produces in
all tissues it comes into contact with as a liquid or as a vapour. It is a potent
irritant that produces a massive inflammation in the skin and the respiratory
tract that culminates in tissue necrosis. Although the skin injury and injuries
to the eyes are rarely fatal, damage caused to the respiratory tract can result in
fatal lung damage and long term breathing difficulties.
L produces similar toxic effects to SM. There are fewer reports of toxicol-
ogy studies available for L than for SM, but those that are published indi-
cate that L acts more quickly than SM with an immediate irritation that SM
does not produce. The skin lesions are associated with more oedema and
tenser blisters than SM but the immediate irritant effect limits the amount of
agent exposure because it will cause the victim to terminate exposure. Stud-
ies in animals indicate that L is not toxic to reproduction at doses lower than
those that cause toxic effects in the parents and that it produces some in
vitro changes characteristic of a mutagen, but does not give positive results
in accepted tests for mutagenicity. There are no formal studies of its car-
cinogenic potential, but since the arsenic it contains is a group 1 carcinogen
it should be treated as though it is carcinogenic until further information
becomes available.
The mechanism by which SM and L cause vesication remains unknown and
because of this they will continue to be the subject of active scientific investiga-
tion. They will also remain CWAs of concern into the foreseeable future.
Acknowledgements
The author would like to acknowledge the many experts who contributed to
his knowledge of SM toxicology in this chapter, in particular Prof. R. L. May-
nard, Prof. T. C. Mars and Prof. R. P. Chilcott. Thanks are also due to those
who helped directly with the preparation of this manuscript Dr Paul Rice,
Mrs Bronwen Jugg, Dr Chris Green, Mr Jon Scawin and Dr Chris Lindsay.
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toxicity (dominant lethal mutation) study of agent Lewisite, Final Report
(9/15/88–6/14/90), National Center for Toxicological Research, Arkan-
sas, USA, 1993.
175. L. B. Sasser, J. A. Cushing, D. R. Kalkwarf, P. W. Mellick and R. L.
Buschbom, Toxicology Studies on Lewisite and Sulfur Mustard Agents:
two-generation reproduction study in rats, PNL-6978, Pacific Northwest
Laboratory, 1989.
Chapter 3
Toxicology of
Organophosphorus Nerve
Agents
Helen Rice*a
a
Chemical, Biological and Radiological Division, Dstl, Porton Down,
Salisbury, SP4 0JQ UK
*E-mail: hrice@dstl.gov.uk
3.1 Introduction
Nerve agents are highly toxic chemicals that act primarily by inhibiting the
enzyme acetylcholinesterase (AChE), and the primary routes of exposure are
ingestion, inhalation and absorption through the skin. Depending on the
route of exposure and amount absorbed, nerve agents inhibit cholinesterases
(ChEs) in both the peripheral and central nervous system, leading to an excess
of the neurotransmitter acetylcholine (ACh) at various sites of action. Muscles,
glands and nerves can become overstimulated by excessive amounts of ACh,
producing a range of toxic effects and ultimately death by loss of respiratory
function. Nerve agents are divided into two main groups, G agents: tabun (GA),
sarin (GB), soman, (GD) and cyclosarin (GF); and V agents: VE, VG, VM, VR and
VX. G agents are more volatile, posing primarily an inhalation hazard, whereas
V agents, typified by VX, are more persistent, less volatile and absorption
through the skin represents a particularly hazardous exposure route.

81
82 Chapter 3
Toxicological investigations carried out for militarily relevant defensive
purposes have focused on the determination of the doses or concentrations
required to produce toxic effects such as lethality, and the ability of various
protective measures to prevent or treat them. The objective of this type of
study is to assess the impact of the agent on the ability of the armed forces
to maintain operational tempo, taking into account the multiple systems of
detection, protection and hazard management used. Operational risk man-
agement uses toxicology to inform the hazard assessment and management
of chemical agent exposures, graded at various levels from negligible to
marginal, through to critical and catastrophic severities.1 In contrast, more
mainstream occupational toxicology is generally aimed at determining “no
observable adverse effect levels” (NOAEL), for the purpose of defining expo-
sure controls.
Defence research relies upon estimates of the effective dose, or dosages, of
agents in humans. For lethal agents, such as the OP nerve agents, these val-
ues cannot be measured or confirmed in humans, so there is a heavy reliance
on extrapolation of animal data to man.2–6 Estimates from animal data can
be supported by the many low dose experimental exposures of human sub-
jects carried out from the 1940s through to the late 1980s. The toxicology of
OP nerve agents has been extensively reviewed in recent years,7–10 but many
of the original reports referred to in these reviews were, until recently, still
classified. Among these reports were those resulting from the programme of
human studies carried out at the Porton Down site in the UK (Figure 3.1)11
that have now been released to the UK National Archives† under the Public
Records Act 1967 and the Freedom of Information Act 2000. The purpose
of this chapter is to review the contents of the declassified reports on stud-
ies of OP nerve agents and place them in context with the existing reviewed
material.
3.2 General Substance Information

3.2.1 Physicochemical Properties
The common nerve agents are liquids at ambient temperature and pres-
sure.12,13 The high volatility of G agents (sarin is one of the most volatile
agents) makes them potent weapons if vaporised and effectively dissemi-
nated. Sarin is readily water soluble, and so are soman and tabun to a more
limited extent. The V agents such as VX are much less volatile, have an oily
consistency and a low evaporative rate, making them in a military context
ideal terrain denial agents. VX is lipophilic and readily penetrates the skin
producing delayed effects. The major physical properties for the most com-
mon chemical warfare nerve agents are summarised in Table 3.1. Nerve
agents possess a unique phosphorus–carbon bond that is not present in pes-
ticides, which are characterised by a P–O or P–S linkage. OP nerve agents
†
http://www.nationalarchives.gov.uk
Toxicology of Organophosphorus Nerve Agents 83
Figure 3.1 The

programme of human studies on nerve agents at Porton sepa-
rated according to agent type. Human studies with nerve agents were
conducted initially with G agents (GA, GB, GD, GE and GF) from 1945
to 1953. Studies were of three types: vapour inhalation; the effect of
vapour on the eyes; and penetration of the skin and clothing by liquid.
These studies mainly sought to understand the effects of G agents on
man, to estimate threshold and harassing dose levels and to provide
results from which lethal doses could be extrapolated. Human studies
conducted with G agents after 1954 were confined to vapour exposures.
From 1958 to 1969 studies were undertaken on penetration of liquid VX
through the skin and clothing, inhalation and absorption of VX vapour
through the skin, and effects of VX vapour on the eyes. Redrawn from
the Porton Historic Survey.11
have an asymmetric phosphorus atom and, therefore, have distinct chiral

enantiomers. The toxicities of the individual enantiomers vary and are a
reflection of the chirality of their biological targets.14,15 Considerable work
has studied AChE inhibitory rates and toxicities of the separate enantiomers,
which can differ greatly.16–24 The work described in this chapter has been per-
formed on racemic mixtures.
3.2.2 History
The first modern OP nerve agents were developed by Gerhard Schrader in the
1930s, whilst working at Wuppertal-Elberfeld in the German Ruhr valley in
an attempt to find an insecticide to control the spread of the polyxena beetle
that was devastating the valley grape vines. Scientists in Schrader’s team first
made tabun (GA), in 1936, and in 1938 discovered sarin (GB). Other agents,
soman (GD), GE and cyclosarin (GF), were researched by the German Army
Ordnance Department during World War II but were not used in that con-
flict. VX was developed in the UK in response to the perceived threat from the
G (“German”) agents.14
84
Table 3.1 Physicochemical
properties of selected nerve agents.a
Nerve agent and
common name GA tabun GB sarin GD soman VX
Structure
Chemical name O-ethyl N,N-dimethyl phos- Isopropyl methylphos- Pinacolyl methylphos- O-ethyl S-diisopropyl-
and synonyms phoramidocyanidate, ethyl phonofluoridate; phonofluoridate; aminomethyl meth-
N,N-dimethylphosphoroam- O-isopropyl methyl- 3,3-dimethyl-n-but- ylphosphonothiolate;
idocyanidate; dimethylami- phosphonofluoridate; 2-yl methylphos- O-ethyl-S-[2(di-isopro-
noethoxyphosphoryl cyanide; phosphonofluoridic phonofluoridate; pylamino)ethyl] methyl
dimethylaminocyanophos- acid, methyl-, 1-meth- 3,3-dimethyl-2-butyl phosphonothioate;
phoric acid ethyl ester; ylethyl ester; phos- methylphosphono- S-(2-diisopropylamino-
cyanodimethylaminoethoxy- phonofluoridic acid, fluridate; 2-buta- ethyl) O-ethyl methyl
phosphine; dimethylaminocy methyl-, isopropyl nol, 3,3-dimethyl-, phosphonothiolate; eth-
anoethoxyphosphine oxide; ester; isopropoxymeth- methylphosphono- yl-S-dimethylaminoethyl
ethyl dimethylaminocyano- ylphosphoryl fluoride; fluoridate; methyl- methylphosphonothiolate;
phosphonate; phosphorami- isopropyl methyl- phosphonofluoridic phosphonothioic acid,
docyanidic acid, dimethyl, fluorophosphonate; acid, 3,3-dimeth- methyl-, S-(2-(diisopro-
ethyl ester; dimethylamidoe- isopropyl methylphos- yl-2-butyl ester; pylamino)ethyl) O-ethyl
thoxyphosphoryl cyanide phonofluoridate; meth- 1,2,2-trimethylpropyl ester; ethyl S-2-diisopro-
ylfluorophosphoric methyphosphonoflu- pylaminoethyl meth-
acid isopropyl ester oridate ylphosphonothiolate;
ethyl-S-diisopropylamino-
ethyl methylthiophospho-
Chapter 3
nate;methylphosphonothioic
acid S-(2-(bis(methylethyl)
amino)ethyl) O-ethyl ester
Toxicology of Organophosphorus Nerve Agents
CAS registry 77-81-6 107-44-8 96-64-0 50782-69-9
number
Physical state Colourless liquid Colourless liquid Colourless liquid Colourless oily liquid (yellow)
(yellow–brown)
Odour Odourless when pure; faintly Odourless Fruity Odourless
fruity
Molecular weight 162.1 140.1 182.2 267.4
Boiling point (°C) 247 147–150 167–198 292–300
Vapour pressure 0.07 2.9 0.3 0.0007
(mm Hg) at 25 °C
Volatility (mg m3) 600 17 000 3900 9–10
at 25 °C
a
Sources: Dstl internal data and ref. 12 and 13.
85
86 Chapter 3
Subsequently, chemical warfare agents have been used in conflict; nota-
bly during the Iran–Iraq war, and in terrorist attacks in Japan, both of which
involved sarin, as well as tabun in the case of Iran–Iraq.25–27 Very recently,
sarin was used in the Syrian conflict, “on a relatively large scale, resulting
in numerous casualties, particularly among civilians and including many
children”.28,29
3.2.3 Uses
The most toxic OP nerve agents have no uses apart from chemical warfare;
they are subject to strict controls under the Chemical Weapons Conven-
tion, which came into force in 1997 and to which the majority of the world’s
nations are signatories.30 Nevertheless, the potential use of such chemicals in
terrorism remains a concern.31–33 Some organophosphates are used as insec-
ticides (e.g. malathion, parathion, chlorpyrifos), but their use has declined
in recent years as safer pesticides such as pyrethroids have become widely
available. Nerve agents act very rapidly and are lethal in mammals at much
lower doses than pesticides. A significant number of cases of poisoning with
OP pesticides occurs each year, primarily in the developing world, resulting
in 200 000–300 000 deaths annually—many through self-poisoning.14,34–36
3.3 Hazard Characterization of Nerve Agents

Chemical warfare nerve agents are OP anti-ChEs that exert their toxic effects
by binding irreversibly to the enzyme AChE (EC 3.1.1.7), the effect of which
is to significantly increase the quantities of the neurotransmitter ACh at the
synaptic cleft of cholinergic synapses. A rapid or large increase in ACh con-
centration in the peripheral and central nervous system leads to a choliner-
gic crisis.7 Exposure to airborne vapour causes signs of poisoning, typically
in order of increasing dose and severity, of miosis, runny nose, chest tight-
ness, dim or blurred vision, tightness of the chest, nausea, vomiting, cramps,
excessive salivation, sweating, drowsiness, confusion, difficulty breathing,
muscular twitching, convulsions, and loss of consciousness.12 This acute
cholinergic crisis may develop within minutes of exposure to a nerve agent,
and the resultant respiratory failure may be fatal if not treated.37 Subsequent
clinical syndromes described as intermediate syndrome (IMS) and organo-
phosphate induced delayed neuropathy (OPIDN) may occur days to weeks
following exposure, but are more comprehensively described in association
with OP pesticide poisoning.38,39
Cases of human exposure to chemical warfare nerve agents are rare; except
for the uses in war and terrorism cited above, and documented accidental
exposures.40–42 In these circumstances, the doses received are generally
unknown, and the documentation and follow-up of medical interventions
received is poor or inconsistent. An exception to this are the controlled
studies conducted in human volunteers on the effects of low doses of nerve
agent.43,44
3.3.1 Acute Effects of Nerve Agent Exposure

The acute toxicity of nerve agents is due to ChE inhibition in the central and
peripheral nervous system. In the blood, nerve agents bind irreversibly to red
blood cell AChE and also to plasma butyrylcholinesterase (BuChE), which
acts initially as a buffer, preventing some of the nerve agent from reaching the
target tissues and organs (central nervous system and neuromuscular junc-
tions). Nerve agents function as a substrate for AChE, effectively preventing
ACh, the normal substrate, from binding. The substrate binding domain on
AChE contains a glutamate and an aromatic tryptophan that interact with the
ammonium group of ACh, together with an amino acid triad that interacts
with the ester group. The hydrolysation of ACh by AChE is extremely rapid,
in contrast to the hydrolysis of a nerve agent–AChE complex.45 The reactions
of sarin with AChE are shown schematically in Figure 3.2. The phosphorus
atom in the nerve agent binds to the serine hydroxyl group in the active site
of the enzyme (phosphorylation; Figure 3.2a and b). Spontaneous dephos-
phorylation, or reactivation of the enzyme, occurs much more slowly, causing
a persistent inhibition of AChE activity and thus leading to a build-up of ACh
at cholinergic synapses (Figure 3.2c). The agents also rapidly phosphorylate a
wide range of serine containing proteins, predominantly hydrolases, includ-
ing BuChE, and carboxylesterases,46 and are rapidly metabolised, generally by
hydrolysis. Inhibited AChE can be reactivated by strong nucleophiles such as
oximes, which are used in the treatment of nerve agent poisoning (see Chapter
1 in Volume 2). After binding to AChE, some G agents undergo a rapid second-
ary de-alkylation of the phosphate group known as ageing (Figure 3.2d). The
half-time of ageing ranges from a few minutes for soman to many hours for
tabun and VX.47–49 Aged inhibited AChE cannot easily be reactivated, essen-
tially making the agent binding irreversible. Nerve agents generally react more
rapidly with BuChE than with AChE,50 although BuChE reacts preferentially
with the less toxic P–R enantiomer of V agents, in contrast to AChE, which
is selectively inhibited by the P–S enantiomer.24 For G agents, BuChE shows
no stereoselectivity, whereas AChE binds preferentially to the more toxic P(S)–
C(R) isomer.51 The activity of ChE enzymes can be measured biochemically rel-
atively simply as a functional marker of nerve agent poisoning and therapeutic
efficacy.52 As BuChE is not a specific target for OP agents, low levels of BuChE
activity may be associated with a range of conditions unconnected with expo-
sure to OP chemicals, including liver or heart disease, and pregnancy. There
is variation of BuChE expression within and between individuals, and genetic
polymorphism exists.53 It is suspected but not proven that people with BuChE
deficiency are more susceptible to the toxicity of OP pesticides, as well as to
anti-Alzheimer’s disease drugs and recreational doses of cocaine.54
Animal data indicate that nerve agents rapidly distribute to the brain, lungs,
heart, diaphragm, kidneys, liver and plasma. The concentrations in all tissues
initially decline rapidly, followed by a second more gradual decline, reflect-
ing metabolism and urinary elimination, respectively.55 The measurement of
the hydrolysis product of sarin [O-isopropyl methylphosphonic acid (IMPA)]
in urine may be used for retrospective detection of exposure, although it is
88 Chapter 3
Figure 3.2 Schematic

representation of the reactions between sarin and AChE. (a)
Sarin and the active site of AChE combine to form an inhibitor–enzyme
intermediate. (b) The fluoride has been lost, leaving a complex of sarin
and AChE. From this state, either (c) spontaneous hydrolysis and res-
toration of function or (d) dealkylation can occur. (c) The ester link in
the phosphonylated AChE has been hydrolysed, the enzyme has reacti-
vated and alkyl methyl phosphonic acid has been formed. (d) The link
between the alkyl group and the phosphorus has been cleaved. This
produces a conformational change that results in the formation of a
very stable agent–enzyme complex that is then resistant to spontaneous
hydrolysis and reactivation by oximes. This is known as “ageing”. The
rate of ageing is dependent on the nature of the alkyl group and is fairly
slow (hours) in the case of sarin and VX, but is very rapid (minutes) in
the case of soman. Figure adapted from Vale et al.45
eliminated within a few days.56 Other biomarkers of exposure are measure-

ment of BuChE adduct and fluoride reactivation of phosphylated binding
sites.56 These methods have the advantage that they can be used over a longer
period than the detection of inhibited AChE or measurement of metabolites.57
3.3.2 Historical Nerve Agent Toxicity Studies

The UK War Office began research into the toxicity of the G agents, initially
tabun (GA), following the capture of nerve agents from Germany in 1945.
Other G agents, including sarin and soman, were tested at Porton Down in
the late 1940s in a variety of animal species by a range of exposure routes,
with the aim of providing a basis for hazard assessment for humans in the
event of these materials being used in warfare.58–64 Human studies with nerve
agents took place between 1945 and 1953, and mainly sought to understand
the effects of G agents in humans, estimate threshold and harassing dose
levels, and provide results from which lethal doses could be extrapolated. By
1949, antidotal research was underway, evaluating atropine sulphate,65 and
toxicology studies moved on to trials exposing human subjects to vapour
inhalation, investigating the effect of vapour on the eyes, and penetration of
the skin and clothing by liquid.66 Human studies that were performed with G
agents after 1954 were confined to vapour work. Most of the studies sought
to understand the impact on military efficiency of G agent poisoning, and
mainly concentrated on eye effects. VX studies ran from 1958 to 1969. Those
involving service volunteers considered the skin penetration of liquid VX and
the effect of VX vapour on the eyes. Studies on the inhalation of VX vapour
were done only with volunteers from the Porton medical staff.
3.3.2.1 Systemic Toxicity Studies

The exceptionally toxic nature of these compounds, particularly sarin and
soman, to a range of mammalian species was evident from the outset, with
LD50s (dose required to produce lethality in 50% of the exposed population)
in the tens of micrograms per kilogram range, see Table 3.2. The extreme
potency of sarin to irreversibly inhibit ChEs was demonstrated in vitro and
in vivo, and the restoration of function in erythrocyte AChE was recognised
to be about 1% per day, reflecting the rate of production of new red blood
cells.44 It was found that for sarin administered subcutaneously to rats, the
lethal dose was proportional to bodyweight, therefore expression of LD50 in
mg kg−1 is appropriate.67 There was no effect of gender on the LD50 of sarin
administered subcutaneously to either rats or guinea pigs.68,69 Subsequent
studies using subcutaneously administered VX produced broadly similar
conclusions that there was no gender effect, but did demonstrate variations
in LD50 with weight (and age), which reinforced the need to select closely
matched experimental groups of animals for toxicity experiments.70 More
recent studies have indicated gender differences for vapour exposures (see
Section 3.3.2.2). Early work on nerve agent toxicity used a variety of animal
species, as can be seen in Tables 3.2 and 3.3, covering the mild to lethal
effects range in animal species, such that these could be compared to exper-
iments covering the mild to moderate effects range in man with the aim of
reliably extrapolating animal data to human toxic effects estimates. The rat
was used extensively for testing nerve agent treatments until the late 1970s,
when it was realised that the rat was unresponsive to treatments that had
been demonstrated to be of benefit in guinea pigs and monkeys (R. H. Inns,
personal communication). Rats and mice are relatively insensitive to nerve
agent poisoning compared with guinea pigs and non-human primates; this
is because rats and mice, unlike guinea pigs, have relatively high levels of
90
Table 3.2 Summary
of early Porton animal toxicity data.a
LD50 (mg kg−1)
Route of
administration Species GA tabun GB sarin GD soman GE GF cyclosarin VX References
Intravenous Rabbit 0.1023 0.0194 0.0202 0.0567 0.024 61,69,73
Cat 0.015 69
Intraperitoneal Mouse 0.89 0.34 0.20 0.54 0.58 0.05 69,73–75
Subcutaneous Rat 0.16 0.108 0.125 0.084 0.11 0.12 62,68–70,76
0.159 0.087 0.084 0.125
0.208 0.116
0.107
Guinea pig 0.13 0.049 0.027 0.093 0.04 68,69,76,77
0.125 0.038
0.124 0.050
0.038
Rabbit 0.5 0.06 0.02 69
Mouse 0.4 0.223 0.12 0.19–0.24 69,76,78
Monkey 0.038 76
Intragastric Guinea pig 13.8 1.63 0.29 0.90 0.83 64 and 73
Rat 2.8 0.58 0.41 0.47 1.53 63 and 73
Percutaneous Goat (depilated) 6.71 0.913 0.177 0.120 0.47 60 and 73
Rabbit (clipped) 11.8 1.36 79 and 80
11.6
Rabbit (depilated) 4.03 1.26 0.61 2.36 0.41 73,79–81
2.36 0.91
2.7
Rat (depilated) 3.95 11 4.88 14.8 1.81 59 and 73
Horse (clipped) (n = 2) >2 73
Chapter 3
a
All data are from Porton technical papers and memoranda as indicated.
Table 3.3 Selected
experimental toxicity parameters for GB and GD derived from probit analysis.a,b
Toxicology of Organophosphorus Nerve Agents

Nerve Route of LD50 (mg kg−1) or Lower Upper
agent Species administration LCt50 (mg min m−3) 95% CL 95% CL Slope (SE) Notes Reference
GB Guinea pig Subcutaneous 0.049 0.048 0.05 8.43 (1.27) Deaths within 48 h 77
GB Guinea pig Inhalation (30 s) 174 N/A N/A N/A Deaths within 24 h 82
GB Guinea pig Inhalation (9 s) 150 N/A N/A N/A Deaths within 24 h
GB Guinea pig Inhalation (6 s) 155 N/A N/A N/A Deaths within 24 h
GB Mouse Inhalation (10 min) 308 N/A N/A N/A 69
GB Mouse Intraperitoneal 0.34 0.31 0.38 6.11 (0.81) Deaths within 48 h 74
GB Rabbit Intravenous 0.0194 0.0092 0.0226 5.337 Deaths within 48 h 61
GB Rabbit Inhalation (10 min) 144 N/A N/A N/A 69
GB Rabbit Percutaneous 1.26 0.61 1.57 5.54 Depilated skin. Deaths 81
within 48 h
GB Rabbit Percutaneous 11.8 9.5 15.2 6.7 Clipped. Deaths within 79
24 h
GB Rabbit Percutaneous 2.7 2 5.7 4.1 Depilated skin. Deaths
within 24 h
GB Rat Inhalation (10 min) 196 184 208 9.36 (1.41) Deaths within 4 days 58
GB Rat Inhalation (1 min) 219 N/A N/A N/A 69
GB Rat Inhalation (30 s) 163 N/A N/A N/A
GB Rat Inhalation (10 s) 135 N/A N/A N/A
GB Rat Inhalation (5 s) 70.8 N/A N/A N/A
GB Rat Inhalation (3 s) 86.7 N/A N/A N/A
GB Rat Inhalation (2 min) 226 n.d. n.d. N/A Deaths within 24 h 82
GB Rat Inhalation (45 s) 205 182 265 N/A Deaths within 24 h
GB Rat Intragastric 0.58 0.53 0.64 8.72 (1.28) Deaths within 24 h 63
GB Rat Percutaneous 11 n.d. n.d. 1.8 (0.89) Depilated skin. Deaths 59
within 4 days
GB Rat Subcutaneous 0.087 0.078 0.092 5.058 Deaths within 4 days 62
(continued)
91
92
Table 3.3 (continued)
Nerve Route of LD50 (mg kg−1) or Lower Upper
agent Species administration LCt50 (mg min m−3) 95% CL 95% CL Slope (SE) Notes Reference
GB Sheep Inhalation (2 min) 218 185 259 Deaths within 24 h 82
GB Sheep Inhalation (10 s) 222 188 246 Deaths within 24 h
GB Sheep Intravenous 0.015–0.02 N/A Deaths within 24 h
GB Rhesus Inhalation (1 min) 99 84 116 Deaths within 24 h
macaque
GB Rhesus Inhalation (10 s) 79 45 147 Deaths within 24 h
macaque
GD Goat Percutaneous 0.177 0.137 0.248 4.905 Depilated skin. Deaths 60
within 48 hours
GD Guinea pig Intragastric 0.29 0.17 0.38 2.5 (0.57) Deaths within 48 h 64
GD Guinea pig Subcutaneous 0.027 0.014 0.038 4.13 (1.52) Deaths within 48 h 77
GD Mouse Intraperitoneal 0.2 0.19 0.22 6.19 (0.82) Deaths within 48 h 74
GD Rabbit Intravenous 0.0202 0.0183 0.0221 5.099 Deaths within 48 h 61
GD Rabbit Percutaneous 0.61 0.44 0.76 5.34 Depilated skin. Deaths 81
within 48 h
GD Rat Inhalation (10 min) 181 168 195 7.25 (1.74) Deaths within 4 days 58
GD Rat Intragastric 0.41 0.32 0.49 8.26 (2.53) Deaths within 24 h 63
GD Rat Percutaneous 4.88 3.65 5.87 3.78 (0.91) Depilated skin. Deaths 59
within 4 days
GD Rat Subcutaneous 0.084 0.081 0.087 5.075 Deaths within 4 days 62
a
All data are from Porton technical papers.
b
N/A: data not available, n.d.: parameter not able to be determined, SE: standard error.
Chapter 3
plasma carboxylesterase, an enzyme known to bind nerve agents in vitro,
acting in vivo as an endogenous bioscavenger.5,71,72 Non-human primates are
recommended for investigating subtle and/or complex behavioural effects of
exposure to nerve agents.9 Ageing rates of soman-inhibited erythrocyte ChEs
are significantly shorter in humans and non-human primates than in rats
and guinea pigs, a factor that must be considered when interpreting experi-
ments of antidotal treatments for nerve agent poisoning.7
3.3.2.2 Vapour Exposures
Many of the early toxicity studies focussed on vapour exposure, as this was
the most relevant route in a military operational context, and considered
either vapour inhalation or the effects of vapour on the eyes. Sarin in vapour
form is absorbed very rapidly through the lungs and eyes, and there is a very
small difference in the doses that produce mild systemic effects and those
that are lethal. For inhalation toxicity studies, the challenge dose is usually
expressed as the Ct, or concentration (C)–time (t) product. The measure of
toxicity is the LCt50 [the product of the vapour concentration (in mg m−3) and
the duration of exposure (in minutes) that will result in the lethality of 50%
of the exposed population].
Initial vapour toxicity studies at Porton used a standard 10 min exposure
time, exposing large numbers of animals to many concentrations of agent
in order to construct statistically robust dose response curves that could
be analysed by the method of Finney84 to give a probit slope and LCt50 (see
Table 3.3).58 The experiments were conducted in a static chamber where
liquid agent was vapourised by being centrifuged off the edge of a disc
rotating at up to several thousand revolutions per second.85 This “spinning
disc” apparatus was continuously improved to ensure uniformity of flow
and reproducibility of conditions, permitting experiments with constant C
and varying t and vice versa. This enabled the relationship between Ct and
lethality to be studied more comprehensively, and revealed the tendency
for the LCt50 value to fall with decreased exposure times. Early reports
indicate that this phenomenon was much debated, with Porton scientists
initially failing to confirm that at very high concentrations and short expo-
sure times there is a deviation of the lethality from that predicted by lin-
ear extrapolation from longer exposures; a phenomenon first described by
Canadian researchers. This was deemed to be due to the time taken at the
start and end of the exposure for the agent concentration to reach a steady
state in the static chamber. An improved design, the “constant flow appa-
ratus” (Figures 3.3 and 3.4) was developed to overcome these limitations
of the previous equipment, and subsequent experiments produced results
broadly similar to the Canadian ones.69,83
More recently, better controlled studies with improved equipment have
confirmed that the inhalation toxicity of sarin does not obey either Haber’s
law or the toxic load variant of it; that is, the LCt50 is not constant over dif-
ferent exposure durations.86 Gender differences, where observed, are not
94
Figure 3.3 Improved
“constant flow” apparatus for exposing animals to nerve agent vapour. Any predetermined quantity of nerve gas
Chapter 3
could be introduced at a constant rate by varying the syringe size and motor speed. The nerve agent droplets were vapourised
from the glass beads by a heated stream of air. The animal chamber was moved in and out of the apparatus and the animals’
heads were maintained in the vapour stream during the exposure. The animal chamber was kept at a constant temperature
of 25 °C. Source: Porton report 2756.83
Figure 3.4 The

improved constant flow apparatus developed for small animal
exposures. This enabled exposures of constant concentrations for
varying times, or varying concentrations for constant times. Chemical
sampling points were located adjacent to the animal chamber. Source:
Porton report 2756.83
necessarily applicable or consistent across all species; in particular, effects

related to oestrus in female subjects are not directly comparable, as humans
and non-human primates have radically different oestrus cycles to rodent
species. Recent studies have shown that for vapour exposures, female rats
were more sensitive to sarin than male rats over a range of exposure concen-
trations and durations.86 This was also the case for miosis caused by exposure
96 Chapter 3
87
to soman and VX vapour. For mini-pigs, however, this gender effect was
reversed, and male mini-pigs were more sensitive to cyclosarin vapour than
female mini-pigs.88
Nerve agents are known from animal experiments to inhibit ChE,89,90 akin
to similar classes of chemicals that were studied during the war.91 Therefore,
whole blood or red blood cell ChE activity was also measured in human sub-
jects in a number of the exposure trials.11 Early experiments that attempted
to use red blood cell ChE inhibition as a diagnostic indicator of nerve agent
poisoning were inconclusive.92 The single exposure to sarin used in a 1949
study by Davies and co-workers92 at a Ct of 6.4 mg min m−3 (t = 2.5 min) pro-
duced miosis, rhinorrhoea and chest tightness in the volunteer subjects but
was not sufficient to depress red cell ChE activity outside the normal range.
The possibility that ChE inhibition is greatly affected by the route of admin-
istration was noted in that report, and indeed a strong dose–response rela-
tionship between orally administered sarin and red blood cell ChE activity
was subsequently reported by US researchers.44 Higher doses of sarin vapour
(15 mg min m−3), which induced pronounced systemic effects, did signifi-
cantly depress red cell ChE activity.69 In parallel animal work, the relation-
ship between the intravenous LD50 and the inhaled LD50 was examined in
rats, sheep, monkeys and guinea pigs in order to provide a basis for the esti-
mation of the inhaled LCt50 of sarin vapour in humans. This estimate was 250
mg min m−3 for a resting 70 kg man.82 In subsequent years, many mathemat-
ical procedures were developed and refined in order to aid the estimation of
human LCt50 values based on animal data (reviewed in Bide et al.93). Models
such as the toxic load model have been derived to describe the relationship
between exposure time and toxicity, in order to describe the dose–response–
time relationship across all toxic effects, not just the LD50. A separate area of
study in the derivation of human toxicity values from animal derived data
concerns the scaling algorithms and methods used for cross-species extrap-
olation.94 For pharmaceutical purposes, the dose conversion is based on a
normalisation to body surface area, but for toxicity of nerve agents by inha-
lation, the appropriate allometric parameter is minute volume : bodyweight
ratio.1,95 Other factors may influence the conversion used, including differ-
ences in the absorption, distribution, metabolism and excretion (ADME)
between species, appropriate species selection and application of a suitable
safety factor. This is a complex area, and is covered in detail in Chapter 9.
In order to assess the effects of nerve agent vapour to the eyes alone, orona-
sal respirators were employed as a protective measure in a series of exposures
to “harassing doses” of sarin vapour in the Ct range of 14–37 mg min m−3
(t = 1.5–2.5 min).96 Subsequently, apparatus was developed to deliver vapour
directly to the eyes of rabbits and humans inside goggles.97 Moylan-Jones
reviewed, in 1973,98 a number of experiments conducted in the 1940s and
1950s to investigate the effects of localised nerve agent vapour on the eye, and
the decrements in performance of militarily relevant tasks that could result
from nerve agent induced miosis (pin-prick pupils). Substantial impairment
of visual function, as a result of miosis, can occur at agent concentrations
that do not result in any systemic effects. Nerve agent vapour that comes
into contact with the corneal surface rapidly dissolves and produces local
AChE inhibition, resulting in spasm of the constrictor muscle of the iris and
severe miosis.97 Local eye effects of a 3.2 mg min m−3 exposure (t = 2 min) to
tabun vapour included lachrymation, dilatation of the bulbar conjunctival
vessels and pupillary constriction within 3 min, followed by irritation and
development of miosis. The visual decrements could persist for several days.
Exposure to higher Cts (14–30 mg min m−3, t = 10 min) produced circumcor-
neal injection and engorgement of the blood vessels of the iris; these signs
became increasingly evident and reached their maximum between 24 and 48 h
after exposure. Blurring of vision and near point deflection was found to be
due to spasm of the ciliary muscles, which also resulted in severe pre-orbital
headache.99
The threshold Ct for miosis from a single sarin exposure was 3.3 mg min
m−3 (t = 40 min) and miosis commenced at 35 min and lasted up to 48 h.66
Following repeated exposures to low Cts of sarin vapour, the same authors
found that miosis was cumulative and aggravated by subsequent exposures;
pain and blurred vision developed after the third or fourth exposure. Other
general symptoms reported from this series of exposures included tightness
of the chest, nasal congestion and rhinorrhoea, and frontal headache. At a
lower dose, when the exposure time was halved to 20 min, the additive effects
were reduced. Parallel studies in rabbits indicated that the concentration
required for threshold signs was similar to humans, but miosis appeared and
resolved more quickly in rabbits.
Although the volatility of VX is significantly lower than that of G agents,
the vapour does present a hazard, being absorbed quite rapidly both through
the skin and the eyes.100,101 Although VX evaporated slowly, due to the high
toxicity very little vapour was required to produce a toxic concentration. VX
vapour was found to be more potent in producing miosis than sarin vapour,
by a factor of 10–25 times, with the additional factor that its persistence could
prolong the likely exposure hazard.97 For example, in rabbits, the ECt50 (dose
to produce 50% decrease of pupil area) was 1.32 mg min m−3 for sarin and
0.04 mg min m−3 for VX; the doses to produce a 90% decrease in pupil size
were 2.71 and 0.23 mg min m−3, respectively. These ECt50 values were calcu-
lated from data across a range of exposure times from 2 to 100 min, but with
an assumption that the Ct relationship was constant over the range of vapour
concentrations used; an assumption that was later disproved (see above).
Extrapolation from these data led to the conclusion that an exposure to VX
vapour for 30 min at 0.004 mg m−3 would produce severe miosis in humans.97
A later analysis of US data also compared rabbit and human data, assuming
a factor of 25 in the potency of VX compared to sarin. Further assuming that
humans are twice as sensitive as rabbit (as was found to be the case for sarin
induced miosis), and using an experimentally determined human no-effect
level for sarin of 0.5 mg min m−3, the estimated no-effect level for VX vapour in
humans is 0.02 mg min m−3,102 and the ECt50 for miosis is 0.09 mg min m−3.1
These values compare reasonably well with the earlier Porton estimate of
98 Chapter 3
0.12 mg min m for severe miosis, following an 8 min exposure.97 Acute
−3
toxicity estimates for lethality following a 2–10 min exposure to VX vapour of

15 mg min m−3 and to sarin of 35 mg min m−3 have been published.1
3.3.2.3 Penetration of Skin and Clothing by Liquid Agents

Liquid sarin can be absorbed rapidly through the skin, eyes or mucous mem-
branes. Due to its high volatility, sarin can evaporate rapidly from the skin
surface, but if the exposure site is occluded, for example by clothing, then
evaporation is minimised and the LD50 is much smaller. It was recognised
in early experiments that chemical depilation of the skin using “barium
sulphide, 25 parts; soap powder, 2 parts; talc, 7 parts and soluble starch, 7
parts made up with 10% glycerine in water to a thin cream … smeared over a
clipped area of the back and allowed to remain for 1 min, after which it was
washed off with warm water”79 rendered rabbits more susceptible to liquid
sarin than when it was applied to clipped skin (see Table 3.2). Depilation
removed a layer of skin as well as the fur, and it was concluded in 1950 that
human toxicity estimates should be based on the results from clipped rather
than depilated animal skin.11,80 It was noted in a summary report in 1950 69
that “…with GB under temperate conditions any contamination greater than
0.2 g on the bare skin would present a serious hazard, and probably prove
fatal, to man.” Cullumbine reported in 1954 79 a large study that was con-
ducted between August 1952 and May 1953 with liquid G agents, the aim
of which was to investigate the relationship in humans between sub-lethal
doses of various G agents and blood ChE inhibition in order to estimate
lethal doses. In all, 396 men were exposed to liquid tabun, sarin, soman or
cyclosarin, with agent applied to bare skin on the arm or to layers of mate-
rial attached to the arm, as illustrated in Figure 3.5. In parallel, rabbits were
exposed to sub-lethal and lethal doses of the agents, which were applied
to clipped or depilated skin, either directly on the skin or onto two layers
of cloth.79 It was apparent from these studies that absorption through the
skin is a very variable route of entry, with a wide range of individual ChE
responses to a given dose of agent. Penetration through depilated rabbit skin
was faster than through clipped skin; comparative toxicities were reported
by Muir and Callaway80 who postulated that on clipped skin more time was
available for evaporation to occur, necessitating a larger applied dose of the
more volatile agents to enable sufficient amounts to penetrate through the
skin to cause toxic effects. They demonstrated that the differences in LD50
through clipped and depilated rabbits was more pronounced for higher vol-
atility agents (sarin and GE) than for the less volatile soman and cyclosarin.
Cullumbine et al. used the ratio of the LD50 to the ChE50 (the dose required
to inhibit whole blood ChE activity by 50%) determined in rabbits and the
ChE50 dose in humans to estimate the human LD50. In the human exposures,
there was considerable variation in the ChE inhibition produced by GB on
bare skin, and although a small number of individuals had high ChE inhi-
bition (>80%), the mean ChE inhibition in the highest dose groups was less
Figure 3.5 Mode

of application of liquid nerve agent onto the arm through serge
or flannel clothing.
than 50%. Therefore, the human ChE50 was predicted by extrapolation from
the available data, using the probit slope from the rabbit ChE50 results. The
estimates of the percutaneous LD50 (bare skin) derived from that study were
1.5–1.7 g per person for sarin, 0.3–0.5 g per person for soman and 0.54 g per
person for cyclosarin.79
100 Chapter 3
During April 1953 one man out of the six who had received a 300 mg dose
of sarin as 30 drops of 10 mg on a layer of serge fixed to the upper fore-
arm was severely poisoned. He was hospitalised, his breathing temporarily
ceased and he suffered convulsions. He recovered some days later. As a result
of this incident, the maximum dose of sarin to be used subsequently was
reduced from 300 to 200 mg. On 6 May 1953, a service volunteer died after
being exposed to 200 mg sarin, which was applied, in 20 drops of 10 mg, on
top of two layers of clothing (one layer of serge underlaid by flannel) fixed
to the man’s skin.11,103 Human studies were suspended in 1953 pending the
results of the enquiry into the fatality and were resumed in 1954 with strin-
gent conditions imposed.
Work started in 1953 on a series of V agents, which were found from animal
studies to be highly toxic through the skin. VX emerged as the prime agent
warranting further study.11 The physicochemical properties of VX combined
with the extremely high toxicity meant that liquid pickup, from contami-
nated surfaces and objects, was perceived as the primary hazard, rather than
inhalation, especially due to the persistence of these low volatility agents in
the environment.15,104,105 Studies of the penetration of VX through the skin
compared animals in vivo, animal skin in vitro and human skin in vitro.106 It
was recognised that estimates of the percutaneous toxicity of VX, in common
with the other liquid agents previously researched, are complicated by the
differing volatilities and spreading characteristics of the liquids, as well as
the varying nature and condition of the skin, the amount applied and the
length of time the agent remained on the skin prior to decontamination;
these confounding factors continue to challenge researchers up to the pres-
ent day.107–109 The work at Porton in the 1950s and 1960s showed that little of
the VX liquid applied to the skin was lost through evaporation. The VX pene-
trated the skin slowly, and typically there was a delay before either ChE inhi-
bition or the onset of toxic signs occurred.110 Impurities or diluents could
accelerate or retard the rate of penetration.111 A limited study using volunteer
medical staff took place in 1958. A small amount (50 µg) of radiolabelled VX
was applied to the forearm of two men. The penetration rate observed was
similar to that predicted from animal and in vitro human skin studies. No
ChE inhibition was observed following this dose of VX.112
3.3.3 Other Effects of Nerve Agent Exposure

There is evidence that mechanisms other than the inhibition of AChE may con-
tribute to the toxic effects of OPs. It is difficult, however, to differentiate those
effects from the secondary effects of AChE inhibition. Sarin can directly inter-
act with muscarinic ACh receptors and may affect neurotransmitters other than
ACh. Other postulated mechanisms are through second messenger systems
including phospholipase C gamma, mitogen-activated protein kinase (MAPK)
and c-Jun N-terminal kinase (JNK).57 Biomarkers of oxidative stress have also
been reported in rats, following concurrent exposure to sarin and the revers-
ible AChE inhibitor pyridostigmine bromide, as well as following malathion
113,114
exposure. Many of these, however, represent general markers of cell stress
that are commonly observed in response to cellular injury by other chemical
insults.115 A series of studies in mice reported immunotoxic effects of sarin
exposure at doses that did not produce biologically significant AChE depres-
sion in erythrocytes. The results also indicate that low doses of sarin are able
to alter the reaction of the immune system 1 week following exposure.116–118
3.3.4 Delayed and Long Term Effects of Nerve Agent Exposure

Following exposure to doses of nerve agent that cause acute cholinergic
effects, there is evidence that some health effects can persist for weeks to
months. These were reported to include fatigue, headache, visual distur-
bances, sleep disturbances and muscle weakness.57 Following the first Gulf
War (1991–1992), much work was conducted to elucidate whether “Gulf War
Syndrome” could be causally attributed to sarin or cyclosarin exposure, and
“limited/suggestive evidence” was found for an association between sarin
exposure and neurological effects.57 An IMS is well documented in 10–68% of
patients exposed to OP pesticides; it is characterised by muscular weakness
and paralysis that can occur 1–4 days following acute poisoning. In severe
cases it can progress to respiratory failure and death.119 IMS has not been
reported following chemical warfare nerve agent exposure.120
OPIDN is a rare, delayed neurotoxic effect that may occur weeks after an
acute toxic exposure. OPIDN, like IMS, is likely to be a concern only when
individuals have been exposed to multiple lethal doses of agent but pro-
tected by antidotal drugs.1 It is thought not to be caused by effects on AChE
but by delayed phosphorylation in nerve tissue of neuropathy target esterase
(NTE).15 NTE has been shown to have a critical function in maintaining axo-
nal integrity through phospholipid deacylation; the principal site of action
is the neuronal endoplasmic reticulum but it has also been detected in axon
terminals in the spinal cord and hippocampus.14 OPIDN is unlikely to be a
significant complication of chemical warfare nerve agent poisoning due to
the extreme toxicity of nerve agents compared with the high levels of inhib-
itor required to affect NTE.120,121 A study reported in 1982 that looked at the
long term or delayed health effects of chemical agents tested on US military
volunteers found no evidence of long term adverse effects, but could not rule
out the possibility that there had been transient health effects.102
Carcinogenicity has never been associated with OP nerve agents, although
definitive studies are lacking. Mutagenicity assays have been performed in
a number of in vitro and in vivo test systems, and a summary review carried
out for the US Army concluded that VX did not induce mutagenic effects,
although there is a lack of appropriate studies.102 There is some evidence that
the nerve agent tabun is weakly mutagenic in vitro.122 Few developmental
toxicity and neurotoxicity studies have been performed with chemical war-
fare nerve agents; they have mostly used pesticides, particularly chlorpyrifos.
Reproductive effects have not been shown to be associated with VX or sarin at
doses below those that produce overt clinical toxicity, although the design of
102 Chapter 3
some of the animal studies was not optimal. The significance of developmen-
tal considerations, and their possible association with the non-cholinergic
effects of OPs, have been reviewed by Costa.123 A dominant lethal study was
undertaken in rats exposed to sarin by either vapour exposure or parenterally
(intraperitoneal injection). There was no evidence of dominant lethal muta-
tions following exposure to sarin by either route at any dose. No evidence of
foetotoxicity or anatomical abnormalities were observed in any offspring.124
3.3.5 Effects of Low Level Nerve Agent Exposure

Toxicity estimates are generally derived to cover a range of endpoints to
include lethality, severe effects, threshold effects and mild effects.1,12 Chem-
ical warfare agent toxicity estimates (see Table 3.4) have primarily been con-
sidered in the context of military operations, and estimates generally apply
only to adult males; gender and age effects would be expected to alter the
toxicity estimates, but insufficient data are available to produce estimates
with any degree of confidence. Likewise, research into antidotal treatment
of nerve agent poisoning has generally concentrated on the ability to protect
exposed individuals from lethal and severely incapacitating effects. Thus,
there is lower confidence in estimates deriving from the assessment of low
level exposures.57 This is due in part to an insufficiently precise definition of
what constitutes a “low level exposure”, which can range from an exposure
that results in mild transient acute effects to one that has no observable acute
effects but may result in an increased risk of developing delayed or long term
effects; in turn, these effects may be physiological or psychological.1 Most
studies of this type have focussed on acute (short duration) exposures, but
long term, low concentration exposures may also produce cumulative detri-
mental effects; considerable uncertainty remains over the effects of repeated
doses that do not produce signs at the time of exposure.125 Studies pertaining
Table 3.4 Modern

human toxicity estimates for nerve agents.a
Lethality (LD50 or LCt50) Severe effects (ED50 or ECt50)
Agent Liquid Inhalation Percutaneous Liquid Inhalation Percuta-
(mg/70 kg (mg min vapour (mg/70 (mg min neous
individual) m−3) (mg min kg indi- m−3) vapour
m−3) vidual) mg min
m−3
GA 1500 70 15 000 900 50 12 000
GB 1700 35 12 000 1000 25 8000
GD 350 35 3000 200 25 2000
VX 5 15 150 2 10 25
a
LD50 or ED50: the dose at which 50% of the exposed population will show a particular effect
[death or severe intoxication (prostration, convulsions or vomiting)], LCt50 or ECt50: dose
expressed as a product of the concentration (C) and the exposure duration (t) required to
produce the effect in 50% of the exposed population. Estimates depend on exposure duration.
Durations: 2–360 min for inhalation and 30–360 min for percutaneous vapour. Estimates are
based on data from a range of animal and human experiments. Data from ref. 1 and 12.
to this have focussed on agricultural exposures, e.g. to OP sheep dips, but
precise exposure levels have not been well controlled or documented.57 Some
studies have taken the lowest observable effect level (LOAEL) as that which
produces a significant decrease in erythrocyte AChE activity and no other
signs of poisoning at the time of exposure. Following the Tokyo terrorist
attack126 and the Gulf War conflict57 there was increased concern over low
level exposures, which prompted additional research to better characterise
dose responses across the full spectrum.
Animal studies have been conducted with various OP agents, including
soman, DFP and sarin, demonstrating that performance decrements in
behavioural tasks and attention deficits can result from low level exposures.
In one study, guinea pigs were given repeated daily injections of sub-lethal
doses of sarin for 2 weeks and a range of biochemical and behavioural end-
points were assessed.127 In animals receiving a dose that did not produce
overt signs of cholinergic poisoning, there was still an approximate 90%
decrease in RBC AChE activity, and measurable neurobehavioural changes.
Another study, in which rats were exposed to continuous low doses of VX via
a miniosmotic pump, showed a dose–response relationship between AChE
inhibition and reduced bodyweight gain at doses that did not produce effects
on physiological parameters such as heart rate and blood pressure.104
Following exposure to doses of nerve agents that do not produce acute
cholinergic signs and symptoms, it is less clear whether long term adverse
health effects may result. In particular, in any exposed population, there may
be individuals who are genetically and/or physiologically more susceptible
than the “average”. Animal studies have shown that transient behavioural
changes, and persistent biochemical changes in the central and peripheral
nervous systems were present following exposures to doses that were at or
below a tenth of the lethal concentration of sarin.57
A number of animal studies have used miosis (reduction in pupil size) as
an endpoint for dose–effect or dose–concentration–effect studies, as miosis
caused by direct vapour exposure to the eyes can occur in the absence of
significant red cell AChE inhibition. van Helden et al.128 studied the LOAEL
of sarin in conscious guinea pigs and marmoset monkeys exposed to sarin
vapour in air, with respect to miosis, using the ratio of pupil and iris diame-
ters. Exposure concentrations were 7.5, 15, 25, 50 or 150 µg m−3 and the expo-
sure times needed to achieve significant miosis were in the range 10–300 min.
The group size for each test animal was one, and for the vehicle treated air
exposed controls there were six guinea pigs and five marmosets. Both vehi-
cle- and pyridostigmine-pretreated animals were used in the experiments. In
vehicle-pretreated guinea pigs and marmosets the pupil size was decreased
significantly at sarin doses (Cts) of 1.8 ± 0.3 and 2.5 ± 0.8 mg min m−3, respec-
tively; in the marmosets the NOAEL for the effect was 7.3 µg m−3 (=nomi-
nal 7.5 µg m−3). Similar figures were obtained for pyridostigmine-pretreated
guinea pigs and marmosets. AChE activity in guinea pigs’ red blood cells was
stated not to be significantly depressed, although it was 10–30% depressed
compared with the controls. This was described as a pilot study. The way the
104 Chapter 3
data are presented in this paper make it very difficult to see whether there
was a dose response. The fact that group size was one for the test groups
makes it difficult to evaluate the significance of these findings. This study
is, therefore, inappropriate for risk assessment purposes; although in broad
terms, the Cts causing miosis are in agreement with those found by other
researchers in other species.
Hulet et al.,88,129 studied the effects of whole body inhalation exposure to
sarin (and cyclosarin) in the Göttingen mini-pig (19 males and 18 females).
There was a single female control (air). The ECt50 for miosis for 1 h expo-
sures to sarin was 0.043 mg m−3 in males and 0.044 mg m−3 in females. A
study conducted in marmosets investigated the effect on an electroenceph-
alogram (EEG) and cognitive behaviour of a single dose of sarin, which pro-
duced ChE inhibition of about 51%. The animals were studied for 15 months
following exposure. This study found that the low dose of sarin produced
no significant changes in EEG and no decrement in cognitive behaviour in
the task used to measure it.130 The committee on Gulf War and Health con-
cluded that “There is inadequate/insufficient evidence to determine whether
an association does or does not exist between exposure to sarin at low doses
insufficient to cause acute cholinergic signs and symptoms and subsequent
long-term adverse neurological health effects.”57
3.4 Human Estimates of Nerve Agent Toxicity

It is difficult to determine the toxicities of OPs from cases of human poison-
ing, since the dose of the OP and often its identity are not known. Addition-
ally, many cases receive medical antidotal treatment, which is likely to affect
the toxicity. Estimates of the human toxicity of OP anti-ChEs are, therefore,
largely derived by extrapolation from data obtained using animals, usually
rodents such as rats and mice.
The current military toxicity estimates for the agents GA, GB, GD, GF, VX
and HD were established in a 1994 US Army report published in 2004,1 from
which some data have been collated into Table 3.4.
Previously, attempts were made to estimate chemical warfare agent tox-
icity for occupational exposure standards relating to demilitarisation oper-
ations.102,131 Studies that have included repeated dosing to animals, i.e.
subchronic dose studies, have generally focussed on doses that produced
measureable inhibition of red cell ChE; arguably, this is a biomarker of expo-
sure rather than an indicator of toxicity, and therefore its selection as an indi-
cator of lowest or no observed adverse effect may increase the uncertainly in
subsequent extrapolations to the human.132
3.5 Summary
The climate in the world today in respect of chemical weapons is very dif-
ferent to that of post-war Britain (1945) when some of the research reported
in this chapter was taking place. At that time, the pressing requirement
for accurate assessment of the hazards facing military personnel attacked
by chemical agents, and the requirement to identify and develop effective
medical countermeasures to these agents, meant that controlled exposures
of human subjects to chemical warfare nerve agents were considered a vital
component of the ongoing research. The body of evidence compiled in that
period of research from both animal and human exposures resulted in esti-
mates of dose ranges for mild and severe effects for a range of nerve agents
by a range of exposure routes. Several methods of extrapolation from animal
to human data were employed; the degree of confidence in the resultant val-
ues was also assessed when arriving at a final toxicity estimate. Early meth-
ods were based on a series of assumptions, including that the inhaled dose of
GB causing >90% inhibition of red cell ChE was the LD50; this was recognised
quite soon to be erroneous.133 Additional assumptions included, the assump-
tion of a constant ratio (across species) between the LD50 of an agent via the
intravenous route and the inhaled route;82 and the assumption of a constant
ratio between the ChE50 and the LD50, which was the preferred method in the
1962 report.133 By that time, however, the confounding factors of exposure
duration, respiratory minute volume (i.e. exercise effects) and wind speed for
vapour exposures, were recognised.97,100,133 This resulted in multiple toxicity
estimates being produced for various exposure conditions, for example the
LCt50 for an exercising human may be a fifth of that at rest,134 and the dose of
GB vapour required to produce a 90% reduction of pupil area in humans was
7.29 mg min m−3 at an airflow of 0.07 m s−1 but at the higher airflow of 2.2 m
s−1 a dose of 3.83 mg min m−3 would suffice.11
The most recently published comprehensive (military) acute toxicity esti-
mates for a range of nerve agents were based on extensive reviews of all avail-
able animal and human data, including data not released into the public
domain. Table 3.5 shows that the modern toxicity estimates are generally
more conservative than those derived in the 1950s and 1960s by the Porton
researchers. This reflects the increased amount of information available on
which to base the estimates, improved mathematical modelling techniques
and a more cautious interpretation of the data. The move from toxicity esti-
mates for offensive use to estimates for defence combined with an under-
standing of how the effective dosage varies with exposure time has also
tended to reduce estimates of toxicity.
As more nations join the Chemical Weapons Convention,135,136 the use of
OP nerve agents by state parties becomes increasingly unlikely; however,
their use by terrorist groups cannot be discounted. The present day research
into chemical weapon nerve agents is mainly focussed on informing the haz-
ards to those personnel who might encounter these materials in the course
of destruction.137,138 Research into the responsiveness of these materials to
medical countermeasures informs suitable medical approaches in case of
accidental poisoning by them. Such studies are reliant on using animals,
since it is not possible to conduct experimental research into nerve agent
exposure on humans. Therefore, it is imperative that the extrapolation from
animal studies into humans is as reliable as possible; to that end, modern
allometric modelling, ADME and physiologically based pharmacokinetic
models are becoming increasingly more sophisticated.
106
Table 3.5 Changing
estimates of human toxicity for sarin from 1950 to the present day.a
GB lethality (LD50 or LCt50) GB severe effects (ED50 or ECt50)
Liquid (mg/70 kg Inhalation Percutaneous Liquid (mg/70 Inhalation Percutaneous vapour
Year individual) (mg min m−3) vapour (mg min m−3) kg individual) (mg min m−3) (mg min m−3) Reference
1950 200 100–200 (>1 min 10 000–15 000 >15 >100 69
exposure)
1954 1500–1700 79
1962 300 (2 min exposure) 133
1962 135 (0.5 min 133
exposure)
2004 1700 35 12 000 1000 25 8000 Table 3.4
2005 36 (2 min exposure) 93
57 (10 min
exposure)
a
All estimates are based on data from a range of animal species and low dose human exposures as detailed in the text. Inhalation estimates assume resting
respiration rates of 15 l min−1.
Chapter 3
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warfare toxins in drinking water, Management of Intentional and Accidental
Water Pollution, ed. G. Dura and F. Simeonova, 2006, vol. 11, pp. 23–41.
132. R. A. Young, D. M. Opresko, A. P. Watson, R. H. Ross, J. King and H.
Choudhury, Deriving toxicity values for organophosphate nerve agents:
a position paper in support of the procedures and rationale for deriv-
ing oral RfDs for chemical warfare nerve agents, Hum. Ecol. Risk Assess.,
1999, 5, 589–634.
133. R. J. Shephard, An estimate of the human L(Ct)50 for GB, CDEE Technical
Note 10, UK Archive Number WO 189/1466, 1962.
134. R. L. Maynard, The physicochemical properties and general toxicology
of chemical warfare agents, in Chemical Warfare Agents: Toxicology and
Treatment, ed. T. C. Marrs, R. L. Maynard and F. R. Sidell, Wiley & Sons,
Chichester, 2007.
135. Organisation for the Prohibition of Chemical Weapons (OPCW) Exec-
utive Council, Joint National Paper by the Russian Federation and the
United States of America – Framework for Elimination of Syrian Chem-
ical Weapons, in Thirty-Third Meeting of the OPCW Executive Council, 20
September 2013 EC-M-33/NAT.1, OPCW, 2013.
136. Organisation for the Prohibition of Chemical Weapons (OPCW) Execu-
tive Council, Decision on Destruction of Syrian Chemical Weapons, in
Thirty-Third Meeting of the OPCW Executive Council, 27 September 2013
EC-M-33/DEC.1, OPCW, 2013.
137. G. Hess, Destroying Syria’s chemical weapons, Chem. Eng. News, 2014,
92, 21.
138. N. Notman, Eliminating Syria’s Chemical Weapons, Chem. World, 2014,
11, 46–50.
Chapter 4
Toxicology and Treatment of

Phosgene Induced Lung Injury
Bronwen Jugg*a
a
Biomedical Sciences Department, Dstl Porton Down,
Salisbury, Wiltshire SP4 OJQ, UK
*E-mail: bjjugg@dstl.gov.uk
4.1 Introduction
The first chemicals used in modern warfare were intended to incapacitate or
kill by damaging the lungs and making it difficult or impossible to breath.
Chlorine was the first of these lung damaging agents to be used in World
War I (WWI), but was soon replaced by phosgene because this gas was more
potent. A large amount of research has been carried out on the pathophysi-
ological effects of phosgene and methods of treating the lung injury it pro-
duces. This research is reviewed in this chapter.
Phosgene gas (carbonyl chloride; COCl2) was first produced by John Davy
in 1812 by exposing equal volumes of carbon monoxide (CO) and chlorine
(Cl2) to sunlight (see Scheme 4.1).1
Davy describes the odour as being different from chlorine and “something
like that which one might imagine would result from the smell of chlorine
combined with that of ammonia, yet more intolerable and suffocating than
chlorine itself, and affecting the eyes in a peculiar manner, producing a rapid
flow of tears and occasioning painful sensations”. He named the chemical

117
118 Chapter 4
Scheme 4.1
“phosgene” from the Greek words for ‘light’ and ‘to produce’ due to the part
played by light in its formation since no other way of producing phosgene
had yet been discovered.1 By WWI, phosgene was produced in bulk by the
reaction of Cl2 and CO in the presence of an activated carbon catalyst.2 In
addition, it may be generated by the thermal decomposition of chlorinated
hydrocarbons such as chloroform and carbon tetrachloride.3
Phosgene is probably best known as the chemical warfare agent that
caused the greatest number of chemical deaths during WWI.4 Today it
remains an important global toxic industrial chemical (TIC), produced in
megatonne quantities (global consumption is estimated as approximately
5 million tonnes per year in 2006 5) for use in the production of dyestuffs,
isocyanates, polycarbonates, insecticides and pharmaceutical chemicals.
In the USA alone, the manufacture of phosgene is estimated at 1 million
tonnes, with the National Institute for Occupational Safety and Health
(NIOSH) estimating more than 10 000 workers involved in its manufacture
and use.3,6 There is therefore the potential for accidental environmental or
occupational exposure, which could result in mass casualties, in addition
to use in warfare. Phosgene is also considered to be a potential terrorist
threat agent due to its availability and ease of synthesis, as well as its high
toxicity. Although restrictions on the storage and supply of phosgene are
markedly reducing the quantities available, the widespread use of mate-
rials prepared using phosgene as an intermediate, such as polyurethanes,
will ensure its continued presence industrially.7 It is estimated that, in the
USA, more than 99.9% of phosgene is used at the facility where it is pro-
duced and incorporated into the production process.8
The toxicology and clinical manifestation of the effects of exposure to phos-
gene have been thoroughly reviewed.4,9–12 This chapter provides an update on
recent studies evaluating its mechanisms of action, and therapeutic inter-
ventions and clinical management strategies for casualties of phosgene poi-
soning. Clinical descriptions from human poisonings (during occupational
exposure or accounts from exposures during warfare) suggest that phosgene
acts directly upon the lung to cause a non-cardiogenic pulmonary oedema
that progresses to an acute lung injury (ALI), following a non-symptomatic
latency period. However, case reports and wartime experiences do not pro-
vide quantitative values for the exposure doses causing poisoning. Even after
decades of research, the mechanisms of action are not fully understood and
there are no proven therapeutic strategies or evidence-based clinical guide-
lines for the management of ALI following phosgene exposure.
Toxicology and Treatment of Phosgene Induced Lung Injury 119
4.2 Properties
Phosgene (molecular weight 98.92 g mol−1) is a colourless gas at standard
ambient temperature and pressure, with a boiling point of 8.2 °C (47 °F).4,13
Phosgene is heavier than air (relative vapour density 3.4). It is slightly soluble
in water and aqueous media, but when dissolved is very rapidly hydrolysed
to form hydrochloric acid (HCl) and carbon dioxide (CO2) with a half-life of
approximately 0.026 seconds (37 °C), this reaction being limited by its low
water solubility (∼0.069 mol l−1; 25 °C; see Scheme 4.2).13–15 Vedder10 and
Prentiss16 both note how the rapidity of the hydrolysis reaction was a prob-
lem during WWI because phosgene could not be used successfully in wet
weather. Production of HCl in phosgene munition shells, due to the presence
of water, was also a problem; the acid attacked the shell walls generating
pressure within the shells.16
4.2.1 Odour
At low concentrations phosgene is said to have an odour similar to that of
newly mown hay, while at higher concentrations a more acrid pungent odour
is noted. Due to the mildly pleasant odour at low concentrations, an exposed
individual may not actively seek to escape before lower respiratory tract dam-
age may have occurred.17 The odour threshold is quoted as between 0.4–1.5
ppm (1.6–6 mg m−3); differences in quoted values may be due to differences
between perception and recognition of the odour.18 Although phosgene’s
odour is a sign that it is present in the atmosphere, the odour is not a suitable
indicator of toxicity since odour is a function of concentration (C; mg m−3)
and not the concentration time product (Ct; exposure dosage; mg min m−3).13
This means that at low concentrations, below the odour threshold, damage
may occur due to prolonged exposure. Olfactory inhibition can also occur
rapidly.13 Table 4.1 shows typical estimated human dose related responses to
acute phosgene exposure.
4.2.2 Pathophysiology
Diller describes three main phases associated with phosgene induced lung
injury as the initial reflex syndrome, the clinical latent phase and the clinical
oedema phase.11
Scheme 4.2
120 Chapter 4
Table 4.1 Correlation

of phosgene concentration and dose effects in humans.a
Concentration:
ppm (mg m−3) Dose: ppm min (mg min m−3) Effect
0.4 (1.6) Odour perception
1.5 (6) Odour recognition
>3 (>12) Throat irritation
>4 (>16) Eye irritation
>4.8 (>19.2) Cough and chest tightness
<25 (<100) “Harmless”
>30 (>120) Beginning of lung damage
>150 (>600) Clinical pulmonary oedema
500 (2000) 50% mortality
a
Conversion factors: to convert concentrations in air (at 25 °C) from ppm to mg m−3: mg m−3
= (ppm) × (molecular weight of the compound)/(24.45). For phosgene: 1 ppm = 4.05 mg m−3.
Compiled from published literature.11,13,18,24
4.2.2.1 Initial Reflex Syndrome

Immediate onset symptoms following phosgene exposure result from the
irritant effects on mucous membranes and may trigger protective reflexes. As
with the odour, the severity of irritant symptoms is related to vapour concen-
tration and not dose. Therefore, low concentrations of phosgene for longer
exposure periods may produce pulmonary oedema without irritation of the
upper respiratory tract (Table 4.1). Diller and Zante quote a concentration
of 3 ppm (12 mg m−3) as having an irritant effect on the nose, throat and
bronchi.18
Protective vagal reflexes may be triggered at higher concentrations (>3
ppm; >12 mg m−3) through interaction with sensory receptors within the
bronchial tree. More recently, the potential for these reflexive interactions to
be triggered through activation of transient receptor potential (TRP) channels
has been hypothesised. TRP channels are expressed throughout the airways
from the nasal mucosa to the alveolar capillary system, as described by Buch
et al.19 Ion channels of the TRP superfamily can be subdivided into sensor
TRP channels (e.g. TRPA1, primarily characterized in chemosensory C-fibres
of the lung) or effector TRP channels (e.g. TRPV4, expressed in non-neuronal
cells, such as lung epithelium and pulmonary vasculature). The former may
be directly activated by oxidising and electrophilic inhaled toxic chemicals,
while the latter may represent a more distal effector of signalling pathways,
promoting a complex signalling network to directly or indirectly regulate pul-
monary blood flow, epithelial integrity and mucociliary clearance.19 Banister
et al. demonstrated that the initial irritant action of high concentrations of
phosgene in dogs and cats (5000 mg m−3) is similar to that of ammonia, sug-
gesting that the same pulmonary receptors are sensitive to both substances.
Both species produced a rapid shallow breathing pattern and an increase in
intrapulmonary resistance due to bronchoconstriction.20
However, as phosgene is relatively hydrophobic with low solubility in
aqueous environments, it may not readily diffuse through the mucus and
epithelium to reach underlying sensory nerve endings of the upper respira-
tory tract. As such, low concentrations may not trigger defensive responses
and exposure may go undetected, allowing deeper penetration to the alve-
olar region of the lungs.21 It might therefore be classed more accurately as
a pulmonary rather than sensory irritant, stimulating C-fibre receptors in
the airways.22 Bessac and Jordt agree with Nash and Pattle who analysed the
penetration of phosgene across moist biological membranes and concluded
that, due to the rapid rate of hydrolysis in aqueous environments, intact
molecular phosgene would only penetrate a few tens of micrometres below
the surface.21,23 They hypothesised that, as a result, the upper respiratory
tract would be significantly protected from the toxic effects by the mucous
surface layer, while the alveolar gas exchange regions would be susceptible
to damage, and concluded that phosgene’s reactivity towards certain biologi-
cally important groups was responsible for the toxic effects of the gas.
The potential inability of the chemosensory system to detect phosgene
may compound the damage inflicted to the respiratory system. Additionally,
hydrophobic chemicals such as phosgene may not readily interact with aque-
ous compounds in the airway fluids, whose function it is to sequester and
neutralise reactive chemicals.21
At very high concentrations (>200 ppm; >800 mg m−3) a very different
picture presents, with signs of poisoning that may include apnoea, bron-
choconstriction, epithelial desquamation and inflammatory changes in the
bronchial region. At these high concentrations phosgene may pass through
the alveolar epithelium of the blood–air barrier, and reach the lung capillar-
ies where it reacts with blood constituents, causing the breakdown of red
blood cells in the circulation and the release of cell contents into the blood.
This results in reduced blood flow; Diller describes how death may occur
within a few minutes from “acute cor pulmonale” (acute overdistension of
the right heart), before the development of pulmonary oedema.11,13
4.2.2.2 Clinical Latent Phase

Following acute exposure, the biochemical effects may be immediate while
the clinical effects may be delayed for many hours, hence few if any ill effects
are experienced during the “clinical latent phase”.11 The duration of the
latent phase is inversely proportional to the inhaled dose and may be a prog-
nostic indicator; the shorter the latency period the worse the prognosis.13,24
Vedder describes how, following a non-symptomatic period, symptoms of
pulmonary oedema may occur, which may be precipitated by physical activ-
ity; even “visiting the latrine” could result in progressive dyspnoea that if not
immediately treated could be fatal: “after exposure to moderate concentra-
tions of phosgene a man may feel able to carry on his work for an hour or two
with only trivial symptoms, but he then becomes suddenly worse, presents
a picture of extreme cyanosis which later passes into a greyish-white stage
of collapse which is rapidly fatal. Thus men who have passed through a gas
attack and seem to have suffered but slightly, have died some hours later
122 Chapter 4
10
upon attempting some bodily effort”. It is likely that the physical exertion
increases the demand for oxygen, thus increasing the stress on the heart and
lungs, and uncovering subclinical effects not seen at rest. This may precip-
itate a fatal outcome due to the ongoing damage to the lungs, not evident
when the individual is asymptomatic.
The exact location of the initial cellular interaction of phosgene within the
respiratory tract during the clinical latent phase remains inconclusive. His-
tological changes (emphysema and sloughing of the bronchiolar mucosa)
within the respiratory bronchioles of dogs “at once” after exposure to moder-
ate (500 mg m−3; 30 min) or high (3000 mg m−3; 3 min) doses of phosgene were
reported by Coman et al.25 This was followed by perivascular and peribron-
chial oedema, which preceded alveolar oedema. The rate of development and
extensiveness of the lesions were approximately proportional to the exposure
dose. Pawlowski and Frosolono reported the earliest (immediate) morpholog-
ical change seen in rats exposed to phosgene (4000 mg min m−3) as vacuoles
in ciliated and Clara cells of the bronchiolar epithelium. This was followed
by extracellular septal oedema with intracellular oedema of the walls of the
respiratory bronchioles by 30 min, supporting Coman et al.11,25,26 On the other
hand, Boyd and Perry reported histological changes of alveolar epithelial cells
at the blood–air barrier as being the initial site of injury in rabbits (270 mg m−3;
30 min).11,27 Differences in opinion as to the initial site of action may be due
to differences between species, exposure techniques/experimental design or
doses of phosgene used in the different studies. Lower doses may cause effects
within the respiratory bronchioles, with higher doses reaching the distal alveo-
lar regions.21,23 By the end of the latent phase, movement of blood plasma from
the interstices to the alveoli may occur, causing pulmonary oedema.
4.2.2.3 Clinical Oedema Phase

The presence of pulmonary oedema in humans is difficult to determine since
few symptoms of poisoning may be evident early on. The clinical oedema
phase only becomes truly apparent when sufficient pulmonary oedema fluid
rises into proximal segments of the respiratory tract for the individual to
experience progressive dyspnoea due to insufficient gas exchange. Conse-
quently, respiratory acidosis [increased partial pressure of carbon dioxide
in arterial blood (PaCO2) and decreased pH due to decreased ventilation]
followed by mixed respiratory and metabolic acidosis (decreased pH due to
increased production of hydrogen ions) may occur. As the alveolar–capillary
blood–air barrier becomes more damaged, the oedema fluid becomes more
proteinaceous and leukocytes migrate into the alveolar interstices. Acute
extravasation of plasma constituents into the alveolus may be associated
with surfactant dysfunction, intra-alveolar accumulation of fibrin and colla-
gen, and increased recruitment and activation of inflammatory cells.5,28 Pul-
monary arterial pressure remains normal until the terminal phase, at which
point the heart rate increases. Death results from anoxaemia (reduced oxy-
gen content in arterial blood).11
Vedder describes two types of pulmonary oedema that can be identified,
dependent on the proportion of O2 and CO2 in blood.10 “Blue” type pulmo-
nary oedema results in asphyxia due to decreased arterial blood oxygenation
and increased CO2 levels; hence, the face appearing blue or deeply cyanosed
due to capillary dilatation. As the patient endeavours to obtain more oxy-
gen there is a considerable increase in respiratory frequency, and cough and
expectoration of large quantities of serous frothy fluid is noted; the pulse rate
remains normal (∼100 beats per min). “Gray” type pulmonary oedema results
in an ashen pallor with rapid shallow respiration and is due to reduced lev-
els of CO2 in the blood due to contraction of superficial capillaries such that
little blood passes through them. There is little cough or expectoration, and
the pulse rate is rapid (130–150 beats per min), with low blood pressure. This
is due to the heart being dilated to the point of inefficiency, and results in a
poor prognosis with most fatal cases passing through this stage and death
seen within the first 24 hours after exposure.
4.3 History of Use

The only human toxicity data that exist tend to be from high level, short term
phosgene exposures derived from wartime experiences and industrial acci-
dents. These case reports rarely provide quantitative values for the phosgene
exposure dose. Fatalities and large scale evacuations due to phosgene release
in the industrial setting remind us that phosgene is one of the most lethal
high production volume chemicals in use today.13
4.3.1 Warfare
Vedder10 and Prentiss16 both provide thought provoking introductions to
the use of gas in warfare, drawing on experiences from WWI. The number
of gas casualties admitted to hospital in France is recorded as 70 552, with
1221 deaths, while the total number of gas casualties (Germany, France,
Great Britain and the USA) was estimated at 507 000.10 Phosgene was first
used by Germany against British troops near Ypres on 19 December 1915,
in combination with chlorine. Eighty-eight tons of gas were released from
some 4000 cylinders laid along the front line. However, some months earlier
the British Intelligence Service had become aware of the proposed attack,
and a new helmet (the P Helmet) had been developed, providing protection
and saving many lives, although on this occasion alone 1069 casualties and
120 fatalities resulted.16 Mixtures of phosgene and chloropicrin were also
used during this conflict, the intention being that the chloropicrin would
cause such intense irritation and vomiting that troops would be compelled
to remove their gas masks, when the lethal effects of phosgene would act.
Vedder quotes the lethal concentrations for a 30 min exposure to chlorine
as 3000 mg m−3; 360 mg m−3 for phosgene; and 800 mg m−3 for chloropic-
rin. Phosgene accounted for some 85% of all deaths attributable to chemical
124 Chapter 4
10,16
weapons during WWI. Of the fatal cases, 80% died within the first 24–48
hours after exposure. Survivors of this initial period generally died as a result
of secondary pneumonia.4
The toxicity of phosgene in humans (LCt50) is often quoted as 3200 mg min m−3;
however, the origin of this figure has not been identified or verified.4 Vedder
describes how as little as 1000 mg m−3 may be lethal to humans if exposure
lasts more than a few minutes, while other estimates from WWI suggest the
2 min lethal concentration, 50% (LC50) value for humans is 3160 mg m−3.10,29
Prentiss quotes 500 mg m−3 as being fatal after a 10 min exposure.16
4.3.2 Occupational/Accidental Exposures

In 1976 the NIOSH reviewed the criteria for a recommended exposure stan-
dard for phosgene.6 The report provides a comprehensive review of occu-
pational exposures to phosgene. In 2004, the American Chemistry Council
Phosgene Panel established a phosgene exposure registry (named the Diller
Registry in honour of Dr Werner F. Diller) designed to collect data from US
phosgene producers, the primary purpose being to monitor the health of
workers with acute exposure.30 It should be noted that exposure doses were
estimated based on colour changes from personal monitoring devices or
badges, which change colour based on dosage of phosgene in ppm min.
Colour changes at levels below 1.0 ppm min of phosgene were observed,
indicating sensitivity. The registry examined self-reported symptoms, up to
48 hours and 30 days after exposure, among 338 workers with phosgene expo-
sure. The average dose among workers was 8.3 ppm min (∼33 mg min m−3),
with most workers (234) exposed to <10 ppm min (40 mg min m−3). The
average duration of exposure was reported as 0.1 min, although two lon-
ger exposure times were reported (90 min). The most commonly reported
symptoms within the first 3 hours of exposure were irritation of the nose,
throat and eyes, as well as coughing, after which most of these symptoms
disappeared. It was found that the dose of phosgene and age of the subject
were significant predictors of the presence of symptoms during the first 48
hours. However, there was no relationship between phosgene exposure and
the presence of symptoms 30 days after exposure. It is likely that the dose of
phosgene absorbed by inhalation during such short exposure periods may be
highly variable, in line with the author’s conclusions that the findings were
consistent with acute symptoms being due to concentration dependent,
transient, irritant effects on mucous membranes of the upper respiratory
tract. The findings support the theory that prolonged respiratory effects do
not occur with doses lower than 150 ppm min (600 mg min m−3).30 Pauluhn et
al. discuss how the data suggest that the majority of US industrial exposures
to phosgene are below the presently accepted American Conference of Gov-
ernmental Industrial Hygienists (ACGIH) occupational exposure standard or
threshold limit value (TLV) of 0.4 mg m−3 (0.1 ppm; 8 hours or ∼200 mg min
m−3); they suggest further information is required to confirm that this level
remains appropriate in the occupational setting.5
Table 4.2 Symptoms of patients following accidental phosgene exposure. a,b
Time Symptom Patients

Immediate Choking like sensation and cough 100%
2–3 hours Ocular symptoms 30%
>3 hours Breathlessness 100%
Diffuse chest pain 50%
Vomiting 50%
On examination Tachypneic 80%
(>11 hours) Hypotensive 60%
Diffuse bilateral coarse crepitations 60%
Arterial blood gas Metabolic acidosis 60%
Leukocytosis 30%
Hyponatraemia 30%
Hypokalaemia 40%
ECG Sinus tachycardia 100%
Treatment NAC (1–10 ml 20% solution or 2–20 ml 10% solution 100%
via nebulisation every 2–6 h)
Intravenous corticosteroids 100%
Mechanical ventilation (hypotensive patients) 60%
a
ECG: electrocardiography.
b
Table compiled from Vaish et al.33
Recent reports of accidental exposures to phosgene suggest a similar pro-

gression of injury, although none provide any estimate of exposure concen-
tration.31–34 Immediate signs of irritation suggest relatively high exposure
concentrations, with early symptoms ranging from sore throat to lacrima-
tion, nausea, dry cough and a burning sensation in the mouth and throat.
Dyspnoea and reduced arterial blood oxygenation status may be evident
from 3 hours, with a progressive deterioration over the course of the next
24 hours. Vaish et al. reviewed a cohort of 10 patients admitted to hospi-
tal after an accidental phosgene exposure in India.33 The exposure dose for
these patients is not known. Table 4.2 describes the time course of symp-
toms pre- and post-hospital admission along with the treatment provided.
Despite treatment with N-acetylcysteine (NAC) and intravenous steroids for
all patients, four patients died having been on the ventilator for >4 days.
Two ventilated patients (<4 days) survived along with four non-ventilated
patients. At the 6 month follow up none of the surviving patients reported
any chronic sequelae.
4.4 Haber’s Law

Haber’s law, or rule, was developed by Fritz Haber, a German chemist, in
the early 1900s to characterise the acute toxicity of chemicals used in gas
warfare.35 It states that identical products of concentration (C) of a gas in
air and duration of exposure (t) will yield an identical biological response,
i.e. it assumes a linear response across all concentration levels, including
126 Chapter 4
sub-lethal effects at low concentrations. Empirically this may be expressed
as eqn (4.1):

C × t = K (4.1)

where C is concentration (mg m−3), t the exposure duration (min) and K is a
constant.
However, animal experiments have demonstrated that this rule does not
always apply and may depend upon the endpoint or toxicological response
being studied. Even where it does apply, it may only be valid over a finite
range of exposure concentrations and times. Evidence within the literature
suggests that, following phosgene exposure, Haber’s law applies within cer-
tain limits.5,11,36 For example, when Rinehart and Hatch studied the effects of
phosgene exposure on pulmonary gas exchange in rats (2–10 mg m−3) for time
intervals between 5 and 480 min, they concluded that “there was no indication
that concentration contributed more than exposure time to the magnitude of
change in pulmonary performance over the range of Ct values studied”.37 In
this experiment, the rule was valid, within the sub-lethal range of concentra-
tions and times used. Boyland et al. examined the relationship between LCt50
and exposure time in rats, mice and guinea pigs.38 Re-analysis of these data
(using the probit analysis method of Finney39) supports the conclusion that,
for small animals, the Ct product is constant at exposure times >10 min. The
increased variation in LCt50 at exposure times <10 min is likely due to the reflex
reduction in breathing caused by phosgene in some individuals, as well as the
increased variability in response at such short exposure times (Figure 4.1),
Figure 4.1 Variation

of LCt50 with exposure time for rats, mice and guinea pigs
(re-analysed from Boyland et al.38). Points are LCt50 ± 95 CL (for those
sets of data that could be analysed) using the probit analysis method of
Finney.39
reducing the inspired dose. Pauluhn suggested that the simple mode of action
of phosgene is consistent with it following Haber’s law as long as the exposure
concentration is low enough not to stimulate vagal C-fibres, thus provoking
changes in ventilation. They identified the concentration of phosgene in rats
at which alterations in minute volume occur as 25 mg m−3.36,40
In order to account for the possible non-linear aspects of the toxicological
response to phosgene, the toxic load model can be used, which is exponen-
tial rather than linear with respect to concentration and therefore has the
flexibility to more closely approximate nonlinear sub-lethal effects at low
concentrations. This may be expressed by eqn (4.2):

Cn × t = K (4.2)

where C is concentration (mg m−3), n the exponential function ≠1, t the
exposure duration (min) and K is a constant.
Pauluhn et al. calculated the toxic load exponent (n) for phosgene in rats
using concentration–mortality curves for 30 (54.5 mg m−3), 60 (31.3 mg m−3)
and 240 min (8.6 mg m−3) exposures as n = 1.1.5 The stimulation of protec-
tive reflexes at high phosgene concentrations, especially in rodents, whose
breathing patterns change during the first few seconds of exposure (respira-
tion transiently decreases thus reducing the inhaled dose36), makes extrap-
olation between exposure durations difficult. As a result, when calculating
the toxic load exponent “n”, 10 min exposure data tend to be excluded as rats
appear more resistant to high exposure concentrations. This rodent specific
response highlights the importance of the animal species selected for exper-
imental studies when looking to extrapolate effects to humans. Due to the
oronasal breathing patterns of dogs and pigs the pulmonary dose of phos-
gene is likely to be more similar to humans in these animals than in obligate
nose-breathing rodents.
Dysregulation and overstimulation of pulmonary C-fibres is suggested as
an essential contributing factor for the lethal effects of phosgene.40 High
exposure concentrations over very short durations may result in early symp-
toms (irritation of mucous membranes) but no long term effects, while lower
concentration exposures over longer durations may show no immediate
symptoms but can precipitate a fatal outcome. Hence, it is paramount to
understand that the absence or presence of initial symptoms is not prognos-
tic of longer term outcomes.24
4.5 Tolerance
Following intermittent, repeated exposures of fixed duration, increased
tolerance to recurrent exposures have been reported. Box and Cullumbine
reported an apparent reduction in susceptibility to phosgene poisoning fol-
lowing prior phosgene exposure in rats exposed to 80 mg m−3 (10 min) and 5
days later exposed to lethal concentrations in the range of 220–440 mg m−3
128 Chapter 4
(10 min). This resulted in a lower mortality (reduced from 74 to 33%) com-
pared with rats exposed to the single lethal exposure concentrations.41 This
differed from studies performed in dogs by Underhill (176–480 mg m−3; 30 min),
who concluded that recovery from gassing increased the likelihood of death
from re-gassing. Underhill explains that tolerance is demonstrable only with
low concentrations and does not decrease subsequent reactions to lethal con-
centrations.42 This is in agreement with the conclusions of Kodavanti et al., who
demonstrated in rats exposed for 6 hours per day to a range of concentration–
time profiles designed to provide equal Cts for all concentrations (4 mg m−3)
at one time point (4 or 12 weeks) that the histological effects of sub-chronic
phosgene exposure were driven by concentration rather than Ct.42,43 This was
further supported by Hatch et al., who reported that exposures that cause
maximal chronic injury (measured as hydroxyproline levels or trichrome stain-
ing for collagen) involve high exposure concentrations (that overwhelm the
adaptive response) and longer times between exposures (adaptation being
reduced with increasing time between exposures), rather than high Cts.44,45
They suggest the lack of Ct correlation may be due to an adaptive response
between exposures. The adaptive response relies upon the lung’s ability to
repair itself (e.g. through antioxidant changes) and that the toxicity is not so
severe as to overwhelm the adaptive changes that may occur between expo-
sures (i.e. it occurs at low concentrations). Adaptive responses may protect
the lungs from longer term chronic injury.
4.6 Mechanisms of Phosgene Injury

The molecular pathology of phosgene was once thought to be exclusively due
to the hydrolysis reaction of phosgene with the moist surfaces of biologi-
cal tissues, resulting in production of HCl. This theory has, however, been
abandoned for a number of reasons; as described by Diller and others.5,11
Phosgene is approximately 800 times more toxic than the equivalent amount
of HCl. When Potts et al.46 attempted to reproduce the toxic effects of phos-
gene using HCl a quite different toxicological response resulted (immediate
distress and rapid death) compared with the response following phosgene
exposure (few initial effects and delayed death).4 Potts compared rat lung
homogenates challenged with phosgene and ketene, a known acylating
agent, and found a similar clinical picture (delayed toxicity to alveolar struc-
tures with death due to pulmonary oedema), supporting the concept that
the lethal action of phosgene is a direct result of acylation reactions and not
production of HCl. Nash and Pattle calculated that at a phosgene concentra-
tion of 100 mg m−3 the amount of HCl that would be produced at the blood–
air barrier (∼1 µm thickness) was negligible (7 × 10−10 M), and certainly not
enough HCl would be produced within the lower airways to overcome the
natural buffering capacity of the lung tissue.23 Therefore, HCl production is
no longer thought to provide a major basis for phosgene induced lung injury.
Phosgene, being a highly reactive (electrophilic) hydrophobic gas, is more
likely to penetrate the lower respiratory tract without marked retention in
the conducting airways, having a direct effect on the respiratory epithelium.
Here it reacts with a wide variety of nucleophilic biological components
through acylation reactions (through its carbonyl group C=O) with primary
and secondary amines (–NH2), hydroxyl (–OH) and sulfhydryl (–SH) groups
causing the downstream release of arachidonic acid mediators such as leu-
kotrienes.47 Damage to the respiratory epithelium causes disruption to the
integrity of the alveolar–capillary blood–air barrier, which can result in the
movement of tissue fluid and inflammatory cells (mainly neutrophils) into
the alveolar space (Figures 4.2 and 4.3). Accumulation of activated neu-
trophils in the lung can cause injury through the release of reactive oxy-
gen species. Phosgene causes upregulation of oxidative response enzymes
(increased glutathione reductase and superoxide dismutase activity48,49) as a
result of the lung’s response to injury as well as direct damage to surfactant
and peroxidation of lipids.11,14,28,50 Phosgene has also been shown to deplete
lung nucleophiles, particularly glutathione (GSH), resulting in a decreased
ratio of the reduced GSH to oxidised [glutathione disulphide (GSSG)].51 Due
to inhibition of several enzymes related to energy metabolism (e.g. ATPase,
lactate dehydrogenase, cytochrome c oxidase4,52), cellular glycolysis and oxy-
gen uptake are decreased following exposure, with a resultant decrease in
Figure 4.2 Lung

morphology from pig lung 24 hours post air exposure. A and B:
alveolar spaces. C and D: bronchiolar conducting airways. Alveolar
spaces (B) were free of inflammatory cell infiltrate and oedema (*). The
lumen of conducting airways (C) were free of mucus and cellular debris
(*) with normal pseudo-stratified columnar epithelium with a brush
cell border (D; ↓). Dark scales are 200 µm and white scales are 50 µm.
Stippled boxes show the area expanded in the adjacent image.
130 Chapter 4
Figure 4.3 Lung

morphology from pig lung 23 hours post phosgene exposure (2500
mg min m−3). Extensive alveolar oedema was evident throughout the
lung tissue (A), which was associated with an acute inflammatory cell
infiltrate (B) consisting of macrophages, neutrophils and lymphocytes
(*). Some conducting airways (C and D) showed oedema and inflamma-
tory cell infiltrate within and beneath the submucosa (*) and areas of
the bronchiolar epithelium (D) did not show the characteristic brush
cell border (↓). Dark scales are 200 µm and white scales are 50 µm. Stip-
pled boxes show the area expanded in the adjacent image.
intracellular ATP and cyclic AMP, associated with increased permeability of

pulmonary vessels and pulmonary oedema.5,13 This loss of enzyme activity
may lead to loss of cellular function and cell death. Figure 4.4 shows a sche-
matic representation of the proposed mechanisms of phosgene induced ALI.
4.7 Therapeutic Research Approaches

The mechanisms of action, especially the initiating events, of phosgene
induced lung injury are still not fully understood, despite decades of research.
As a result, there are still no proven therapeutic strategies or evidence based
guidelines for the management of this injury.24,53 Generally, management
of patients is supportive, requiring an intensive care environment. This
approach may be applicable in small scale releases with relatively few casual-
ties, but is unlikely to be an effective means of dealing with larger numbers of
patients following large scale industrial or deliberate terrorist release. Con-
tinuing research is required to better understand the mechanisms of phos-
gene induced injury in order to develop novel therapeutic interventions with
wider applicability.

representation of the proposed mechanisms of phosgene
induced ALI.
There are many reviews on the therapeutic management of phosgene

patients4,24,51,54 thus it is not the intention to reproduce these here. Suffice to say
that the mainstay of therapy, as first described by Vedder, is rest and oxygen.10
This has remained the case since WWI, and although research into the thera-
peutic management of these patients has been ongoing through the decades,
no true advances have been made that have changed this approach, other than
the use of protective ventilation strategies within an intensive care environ-
ment. This section discusses some of the most recent and promising studies
assessing mechanisms of action and therapeutic options in a range of models.
4.7.1 In vitro Studies

The complexity of living organisms can be a barrier to the identification of spe-
cific biological interactions. However, using in vitro techniques it is possible to
simplify the system in order to examine interactions at the cellular or molec-
ular level, allowing detailed examination of specific parts of the system of
interest in highly controllable experiments. Examples include the assessment
132 Chapter 4
of biochemical and physiological processes that may provide information on
mechanisms of action or toxicological end points, against which a therapy
may be targeted. A further advantage of in vitro techniques is that human cells
and tissues, such as human bronchial epithelial cells derived directly from
surgical tissue, allow direct extrapolation to man. The use of in vitro tech-
niques as an alternative to the use of live animals is also widely supported
as the pressure to reduce the numbers of animals used in scientific research
continues to grow. Use of animal models of chemically induced lung injury to
investigate the mechanisms of action and treatment strategies for lung injury
can be highly complex and therefore a way in which more basic mechanistic
research could be performed, as well as the ability to screen potentially ther-
apeutic drugs, would be of benefit. Ideally this new approach should refine,
reduce or replace the use of animals in experimental studies.
Inhalation of phosgene can result in loss of integrity of the respiratory epi-
thelium lining of the lungs. Loss of the integrity of this barrier can result in
movement of tissue fluid into the alveolar space causing pulmonary oedema,
respiratory compromise and death. Effective treatments for such injury need
to be identified and assessed, including a more detailed assessment of the
respiratory epithelium itself. Until recently, real time measurement of the
lung’s epithelial barrier integrity was not possible without the submersion of
cells; this prevents exposure to gaseous chemicals such as phosgene, which
may react with the culture medium to form toxic acids and thus limit the
usefulness of such exposure systems.55 More importantly, for convenience
many cell culture systems are derived from immortalised cell lines, which
can result in an immature and atypical phenotype.56 Use of more phenotypi-
cally normal, primary cells will more closely approximate the in vivo system.
Fully differentiated respiratory epithelium can be achieved using an air–
liquid interface culture system,57 which, in conjunction with the ability to
expose cells to toxic gases and vapours, allows rapid, accurate and cost effec-
tive assessment of a large number of therapies for the treatment of chem-
ically induced lung injury. This may reduce the time to identify promising
therapies with only successful candidates needing to be tested in animals.
Using an air–liquid interface in vitro exposure system, human bronchial
epithelial cells were exposed to phosgene whilst simultaneously monitoring
epithelial integrity (i.e. the functional state of the epithelial barrier) using a
novel on-chip bio-impedance assay that allowed continuous real time mea-
surement of lung cell barrier function.58,59 Measurements of trans-epithelial
resistance were also recorded. Cells were exposed to phosgene at concen-
trations that gave final exposure dosages between 0 and 900 mg min m−3.
The phosgene exposure duration was 20 min. This study demonstrated that
exposure to phosgene caused an immediate, dose dependent fall in electri-
cal impedance compared with air controls, indicating loss of integrity of the
intercellular tight junctions between epithelial cells and hence a breakdown
in barrier function. This loss of integrity of the tight junctions may be an
initiating event in the development of injury following phosgene exposure,
giving a route by which tissue fluid may leak into the alveolar space.
60
More recently, Wijte et al. studied the toxic effects of phosgene exposure
of human lung epithelial cells (A549 human carcinoma cell line) to deter-
mine whether a dose–response relationship demonstrating a clinical latency
period was present in vitro—whether Haber’s law was valid in vitro—and to
identify early markers that may emerge during the latency period and might
provide potential therapeutic targets. Using a CULTEX® exposure system
(platform for in vitro assessment of gases at the air–liquid interface) they
demonstrated dose response effects with exposure concentrations ranging
from 2 to 8 mg m−3 (20 min) for some markers of toxicity, with increased lac-
tate dehydrogenase (LDH, marker of cell membrane integrity) and decreased
mitochondrial activity, in line with decreased cell viability due to phosgene.
Heme-oxygenase (HO)-1 was reduced at 2 and 4 mg m−3, but not at 8 mg m−3,
while intracellular GSH was significantly elevated at 2 mg m−3 and reduced
at 8 mg m−3. In agreement with studies in vivo, downregulation of the anti-
oxidants HO-1 and intracellular GSH demonstrates oxidative stress as a
response to phosgene exposure and supports the use of antioxidants follow-
ing phosgene exposure.13,61 The authors were also able to demonstrate the
validity of Haber’s law using a range of concentration versus time profiles,
resulting in Ct = 160 mg min m−3 for 5 min ≤ t ≤ 20 min. This is in line with
animal studies demonstrating Haber’s law to be valid within certain limits.37
In agreement with our own air–liquid interface studies, and using similar
exposure dosages, Witje et al. were unable to demonstrate a latency period
in vitro, stating that this may be due to the high phosgene exposure dosage
used or because in vitro repair mechanisms may be lacking due to the lack of
a variety of different cell types compared with in vivo studies; thus highlight-
ing one of the disadvantages of in vitro studies.
Cowan et al. exposed human lung small airway cell (SAC) cultures to phos-
gene at 0.4–25.6 mg min m−3 (1 min) and demonstrated dose dependent
increases in IL-8 (neutrophil chemotactic factor) at 24 hours at all but the
lowest dose.62 However, higher exposures (>12.8 mg min m−3) decreased IL-8
from maximal levels; this is purported to be due to increased cytotoxicity, as
measured by trypan blue exclusion, at high exposure dosages. The authors
propose the use of IL-8 as a biomarker both for inflammation following phos-
gene exposure and to assess the efficacy of medical countermeasures in vitro.
These data indicate that exposure to phosgene in vitro induces a rapid cel-
lular response at the level of the epithelium that may indicate one of the
primary injurious mechanisms leading to damage and triggering a series of
downstream events that lead to the delayed clinical picture. The complex
aetiology of phosgene induced lung injury is known to involve a number of
cellular targets that contribute to the injury through a coordinated response
from many cell types both in the lung and in other body systems.63 These
interactions with other organ systems are involved in the development of
the lung injury and may provide novel treatment opportunities. The complex
response of the organism to lung injury is, therefore, difficult to recreate in
vitro and must be studied in vivo if a complete assessment of the injury mech-
anism and response is to be investigated. A major disadvantage of studies
134 Chapter 4
in vitro is in the extrapolation of data back to the whole organ or body system.
Single cell type monolayer cultures exposed at the air–liquid interface have
been used to investigate aspects of cellular injury, and have provided infor-
mation regarding early biomarkers that might be used in the development of
therapeutic options.60 While such models can be used to investigate specific
mechanisms, they cannot model the complex interactions that occur in vivo
during the injury and repair phases of the injury. However, in vitro models are
an important tool that can be used to investigate mechanistic interactions
and to select potential treatment options for further investigation in vivo. By
increasing the complexity of the in vitro systems we can begin to bridge the
gap between simple single cell systems and the whole organism.
Recently, researchers at RTI International and the University of North Car-
olina (USA) have developed a new lung on a chip microfluidic model of the
human airway using primary human cell types (airway epithelial cells, lung
fibroblasts and microvascular endothelial cells) cultured at an air–liquid
interface.64 This model aims to improve the physiological relevance of in vitro
cell culture systems to more accurately predict the effects of, for example,
toxic gas exposure on the human lung. The model could also provide insight
into biological and pathophysiological effects that conventional cell cultures
or animal models do not capture, ultimately leading to the development of
new therapeutic options for lung damage.
4.7.2 Small Animal In vivo Studies

Traditionally, in vivo research has relied on developing a hypothesis of the
injury mechanism, e.g. change in energy metabolism,65 disruption of GSH
redox cycle51,66 and release of arachidonic acid metabolites,47,67 and then
targeting the therapy against the proposed/hypothesised pathway. Multiple
small animal studies have examined treatments for phosgene induced lung
injury with many seeking to provide therapeutic benefit by targeting pro-
posed mechanistic pathways, e.g. the inflammatory response to injury.24,54
However, many of these studies assess pre-treatment options, not viable
within the civilian context, or clinically improbable doses, such that extrap-
olation to humans is unreliable.24 A further issue when investigating thera-
peutic options is the timing of administration of the therapeutic compound.
As the alveolar epithelium deteriorates and function is lost, its responsiveness
to therapy may become increasingly refractive to treatment with increased
time post exposure.68 Administration of inhaled therapies may also become
compromised with time as the lungs fill with oedema.
Liu et al. assessed a single administration of high dose dexamethasone
30 min after exposure of rats to phosgene (1000 mg min m−3).69 Dexameth-
asone treatment resulted in increased mortality and increased measures of
lung injury. Luo et al. further investigated the use of steroids in rats treated
with budesonide, mometasone or dexamethasone 30 min post phosgene
exposure (900 mg min m−3).70 Mortality again increased in the steroid treated
animals, with other measures of phosgene induced injury being worsened.
Both studies used exhaled nitric oxide (eNO) and carbon dioxide (eCO2) as a
non-invasive means of assessing injury. The authors discuss how the lack of
efficacy following steroid treatment along with fall in eCO2 seen following
exposure identifies the initiating events of phosgene induced lung injury as
being caused by dysfunctional alveolar perfusion due to stagnant blood and
haemoconcentration, and not direct tissue injury and acute inflammation.
Endogenous nitric oxide (NO) is an important mediator of inflammatory
responses in the lung and is also involved in neural bronchodilator and vaso-
dilator mechanisms in the regulation of airway and pulmonary blood flow, and
in the pathophysiology of several lung diseases.68,71 Li et al. investigated the
administration of inhaled NO (1.5 ppm for 6 hours started 5 min post phos-
gene exposure, 880 mg m−3; 20 min) in an attempt to ameliorate the effects
of phosgene induced oedema formation in rats. They also investigated the
effects of NG-nitro-l-arginine methyl ester (l-NAME), a non-specific nitric oxide
synthase inhibitor that inhibits endogenous NO production. Inhaled NO pro-
vided no benefit against phosgene injury; in fact, it had a tendency to aggravate
oedema formation. l-NAME administration attenuated the injury as demon-
strated by decreased protein levels in the bronchoalveolar lavage fluid. Pro-
phylactic administration of ethyl pyruvate, an anti-inflammatory compound
with antioxidant properties was also investigated and shown to aggravate
phosgene injury.68 However, Chen et al. showed a protective role for ethyl pyru-
vate in rats exposed to phosgene (1600 mg m−3; 1 min). Ethyl pyruvate was
shown to reduce expression of cyclooxygenase-2 (COX-2) and inducible nitric
oxide synthase (iNOS), resulting in decreased production of prostaglandin E2
and NO, respectively. The authors suggest that this protective effect was medi-
ated in part by inhibition of mitogen activated protein kinase (MAPK) acti-
vation.71 Wang et al. demonstrated increased p38MAPK and translocation of
nuclear factor kappa-light chain enhancer of activated B cells (NF-κB; a pro-
tein complex that controls transcription of DNA) to the cell nucleus in rats,
demonstrating activation of inflammatory and oxidative stress responses fol-
lowing phosgene exposure.72 Ethyl pyruvate has also been shown to induce
HO-1 through a p38MAPK—and nuclear factor erythroid 2-related factor
(Nrf2)–dependent pathway, demonstrating its antioxidant potential.73
Ji et al. investigated the role of Nrf2 in phosgene induced lung injury in
rats (2000 mg m−3; 1 min). Nrf2 is a critical transcription factor known to reg-
ulate multiple antioxidant proteins, including phase 2 detoxifying enzymes,
GSH generating enzymes and the proteasome system, and it has been shown
to protect cells from oxidative stress by promoting GSH synthesis or main-
taining GSH redox status.74 Rats killed 3 hours post phosgene exposure had
mRNA and protein levels of Nrf2 decreased by more than 90%. However,
levels were increased in a dose dependent manner following treatment with
the antioxidant thiol compound NAC. Administration of NAC significantly
elevated lung GSH content and the GSH : GSSG ratio compared with phos-
gene exposed control animals. The authors suggest that Nrf2 is a key medi-
ator connecting phosgene induced lung injury with GSH metabolism, and
that downregulation of Nrf2 expression by phosgene attenuates the ability of
136 Chapter 4
tissues to scavenge free radicals and avoid subsequent oxidative stress. NAC
effectively reversed these effects, as demonstrated by a decreased lung wet
weight to dry weight ratio (an indicator of pulmonary oedema fluid forma-
tion) and oxidative stress markers.
Prophylactic parenteral or oral administration of nucleophiles has pre-
viously been shown to provide protection against phosgene induced lung
injury, providing indirect evidence for the proposed acylation mechanism of
phosgene.75 Pauluhn and Hai described studies using instant high dose aero-
sol exposures to three reactive nucleophiles, hexamethylenetetramine, cys-
teine and GSH, following phosgene exposure in rats (600 mg m−3; 1.5 min).53
Exposure to aerosolised drugs began immediately post exposure and was
continued for 5 or 15 min. None of the treatment regimens studied was asso-
ciated with any improvement in outcome, despite the use of optimised aero-
solisation techniques. This may be due to difficulties in delivering the drug
to the pulmonary regions of the lung in large enough doses to provide protec-
tion (alveolar doses were assessed as being similar to 10 times lower than the
inhaled dose). The authors suggest that phosgene injury occurs immediately
on exposure (as also suggested by in vitro studies discussed previously), with
instant, non-reversible acylation of nucleophilic biological components.53
There may be a lack of secondary responses (downstream changes in bio-
chemical mediators and pathways) to injury at these very early time points.
They suggest that the beneficial effects described by Ji et al., using NAC as a
pro-drug for GSH, are due to beneficial effects on the secondary responses to
injury and not the primary pathophysiological mechanism (instant acylation
of nucleophilic moieties at the initial site of action).
4.7.3 Large Animal In vivo Studies

Studies in small animals have provided mechanistic information on bio-
chemical and pathophysiological pathways following phosgene exposure, as
well as preliminary evaluation of drug treatment effectiveness, but there can
be inherent issues with these models.24 Owing to their small body size and
variations in physiology, small animals are not always the best models to
extrapolate to man in the evaluation of lung injury or therapeutic efficacy.
In comparison, large animals, such as the pig, offer several advantages. The
physiology of pig lung is more comparable to humans than that of rodents,
and their large size and similarities in the tracheobronchial tree and vascular
architectures means that human intensive care unit (ICU) equipment can
be used, allowing greater confidence in the extrapolation of data to humans
than is possible from small animals. The comparative anatomy of the lung
and upper airways of pigs and humans is described by Brown et al.76 The
greatest interspecies difference is in the anatomy of the upper airways, with
pigs having a more highly developed olfactory system than humans. Thus,
pigs have a much greater surface area in the upper respiratory tract, which
may result in greater absorption of inhaled chemicals. Therefore, use of an
animal in which the upper respiratory tract can be bypassed may more accu-
rately reflect human inhalational injury.
A number of studies assessing the efficacy of commercially available treat-
ments that may be available for use by emergency first responders or by buddy
aid prior to medical evacuation were performed in a pig model of phosgene
induced lung injury. The therapies were chosen to act on biochemical and
physiological pathways known to be affected by phosgene exposure, e.g.
inflammation and changes to epithelial barrier function that result in move-
ment of fluid and cells into the lungs.11,24 Treatment studied have included
intravenous and inhaled corticosteroids,77 inhaled salbutamol78 and inhaled
furosemide (Table 4.3).13,79 The studies were performed against a challenge
Table 4.3 Summary

of therapeutic studies using commercially available products
performed in pigs.
Study protocol Study outcome
Evaluation of steroid (methylprednisolone or budesonide) treatment for phosgene
induced lung injury77
Study 1: single intra- Study 2: multiple
venous dose inhaled doses
Group 1: Phosgene + glucose Phosgene + Neither steroid had an
phosgene saline (n = 6) glucose saline effect on mortality, lung
controls (n = 6) oedema, or shunt frac-
tion. However, some ben-
eficial effects on cardiac
parameters, e.g. stroke
volume, left ventricular
stroke work, were noted
Group 2: Phosgene + methyl- Phosgene + Steroids were neither bene-
phosgene prednisolone budesonide ficial nor detrimental in
+ steroid (12.5 mg kg−1) (n = 7) (1 mg per the treatment of phos-
dose) (n = 5) gene induced lung injury

Evaluation of β-agonist (salbutamol) treatment for phosgene induced lung injury78
Group 1: phosgene Saline (0.9%): multiple Salbutamol treatment had
controls (n = 6) inhaled doses no effect on mortality
Group 2: phosgene + Salbutamol: multiple inhaled and had a deleterious
β-agonist (n = 6) doses (2.5 mg per dose) effect on arterial oxygen-
ation, shunt fraction and
heart rate. There was
a significant reduction
in lavage fluid neutro-
phil number and small
decreases in inflamma-
tory mediators in lavage
fluid but not plasma

Evaluation of inhaled diuretic (furosemide) treatment for phosgene induced lung injury79
Group 1: phosgene Saline (0.9%): multiple Furosemide treatment had
controls (n = 8) inhaled doses no effect on mortality and
Group 2: phosgene + Furosemide: multiple inhaled had a deleterious effect
furosemide (n = 8) doses (40 mg) on the PaO2 : FiO2 ratio.
All other measures were
unaffected by treatment
138 Chapter 4
dosage of phosgene that produced a moderate to severe ALI at 24 hours post
exposure, in terminally anaesthetised, ventilated pigs.76,80
Normal human therapeutic doses of steroids were administered to pigs in
a clinically realistic time frame following phosgene exposure (Table 4.3).77
Methylprednisolone (12.5 mg kg−1; human dose equivalent 1000 mg) was
administered intravenously 6 hours post exposure, a time when symptoms
may have developed. Budesonide was administered by the inhaled route
before symptoms presented (multiple inhaled doses from 1 hour post expo-
sure). Neither of these treatment strategies had an effect on reducing pulmo-
nary oedema or mortality, the primary outcomes for the study (in agreement
with Liu et al.69). There were some apparent protective effects on cardiac func-
tion (improved stroke volume and left ventricular stroke work), although the
precise mechanism of this protective effect is not known.77 However, whilst
steroids did not improve outcome they did not cause any detriment either,
and the likelihood is that they will be given in the event of a potential chem-
ical exposure. The current recommended treatment for phosgene poisoning
is 700–1000 mg methylprednisolone or its equivalent, given intravenously in
single or divided doses during the first day and then tapered over the dura-
tion of the clinical illness.77 The use of steroids to treat phosgene poisoning
was based on some of their known actions in decreasing cytokine production
and release from a variety of cell types, and decreasing vascular leakage. De
Lange and Meulenbelt questioned the role of corticosteroids in reducing lung
injury following exposure to chemical agents.81 Their conclusions agree with
our own studies in pigs and rats,69,70 suggesting no beneficial effects from
administration of steroids following phosgene. They propose that potentially
harmful effects may be due to inhibition of repair mechanisms (e.g. impaired
differentiation of type 2 pneumocytes into type 1 alveolar cells).
The β-agonist salbutamol, in addition to acting as a bronchodilator,
increases alveolar fluid clearance by upregulating apical sodium and chlo-
ride channels on alveolar type 2 pneumocytes. It may also reduce alveolar
capillary permeability and, thus, reduce alveolar fluid formation.82–84 Other
known actions include its ability to reduce neutrophil influx into the lung,
blocking inflammatory cytokine release and improving lung mechanics fol-
lowing ALI.85 All of these mechanisms are important in phosgene induced
lung injury, and any amelioration of them may improve outcomes.
Administration of multiple nebulised doses of salbutamol (2.5 mg per
dose) to phosgene exposed pigs using a clinically realistic dose–time regi-
men (Table 4.3; human equivalent 4 mg per dose) resulted in no beneficial
effect on mortality or pathology and worsened various physiological param-
eters including partial pressure of oxygen in arterial blood (PaO2) and shunt
fraction (a measure of the proportion of blood that passes through the lungs
but remains unoxygenated). However, salbutamol treatment reduced neutro-
phil influx into the lung.78
Nebulised furosemide has multiple actions in the lung, including inhibi-
tion of inflammatory cytokine production, induction of prostaglandin syn-
thesis, and prevention of bronchoconstriction and its purported antioxidant
effects, which may be of benefit in the treatment of phosgene induced lung
injury.24,79 Administration of multiple nebulised doses of furosemide to
phosgene exposed pigs using a clinically realistic dose–time regimen (40
mg; Table 4.3) showed no improvement in survival or blood oxygenation,
no change in inflammatory cell influx, and had a deleterious effect on the
ratio of PaO2 to fraction of inspired oxygen (FiO2), a clinical index used in the
assessment of acute respiratory distress syndrome (ARDS).79
The studies described were designed to assess the efficacy of commercially
available drugs against specific biochemical pathways known to be affected
by phosgene exposure. A number of small animal studies have shown ben-
efit when similar pathways have been targeted.24 However, administration
of single treatments for phosgene induced lung injury in our model, using
clinically relevant doses and time scales, did not improve survival or arterial
blood oxygenation. The data suggest that extrapolation from small animals
to large animals and on to human therapy remains a problem, and there
continues to be a lack of evidence based clinical guidelines for treatment of
this injury.24 Combination approaches were not investigated but may pro-
vide a higher likelihood of success, especially due to the complex nature of
the injury and the known redundancy within immune responses. This would
also provide a more realistic investigation of the management of phosgene
exposed individuals in a clinical setting, where single treatments will not be
used in isolation.
The same pig model was used to assess supportive care strategies avail-
able in the ICU, investigating the optimal time for delivering these treatment
strategies. As previously discussed, oxygen therapy has long been advocated
as the mainstay in the treatment of phosgene exposed casualties. Vedder
indicated of the use of 2–10 l min−1 of 100% oxygen in casualties with evi-
dence of pulmonary oedema and marked anoxaemia and cyanosis, for as
long as 1–2 days post exposure. Following this, flow should be reduced to
60% and finally 40% due to the potential toxicity of oxygen itself.10 In 2000,
the National Heart, Lung and Blood Institute Acute Respiratory Distress Syn-
drome Network (ARDSNet) provided the first evidence for improved survival
using protective ventilation strategies in patients with ALI/ARDS.86 The pro-
tocol was based on low tidal volume (TV) and high positive end expiratory
pressure (PEEP) maintaining an oxygenation goal of PaO2 55–80 mm Hg or
oxygen saturation (SpO2) of 88–95%, using incremental increases in FiO2/
PEEP to achieve this goal.
The use of protective ventilation strategies based on the ARDSNet proto-
col were investigated in terminally anaesthetised pigs exposed to a dosage
of phosgene that caused 70% mortality in control animals.87 Anaesthetised
pigs were instrumented, exposed to phosgene and conventionally ventilated
[intermittent positive pressure ventilation (IPPV); TV = 10 ml kg−1, PEEP = 3
cm H2O, 20 breaths min−1, FiO2 = 0.24]. At 6 hours post exposure (to mimic
time for clinical signs to become apparent and movement of the casualty to
an intensive care environment) pigs were randomised to receive either con-
ventional ventilation (controls) or one of two protective ventilation strategies
140 Chapter 4
−1 −1
(TV = 8 or 6 ml kg , PEEP = 8 cm H2O, 20 or 25 breaths min , FiO2 = 0.4).
These parameters, once set, remained unchanged to the end of the study at
24 hours post exposure.
Both protective ventilation strategies significantly improved survival (from
30 to 100% at 24 hours), arterial blood oxygenation and pathology as deter-
mined by reduced haemorrhage, neutrophil infiltration and intra-alveolar
oedema formation (Figure 4.5).87
The effects of oxygen supplementation were also investigated.88 The study
was designed to assess the potential benefit of immediate or delayed oxygen
administration as well as low (40%) versus high flow (80%) oxygen (reflecting
the potential for limited supplies in a mass casualty event). Anaesthetised
pigs were instrumented, exposed to phosgene and conventionally ventilated.
Treated groups received increased FiO2 as follows: FiO2 = 0.80 immediately
post exposure; FiO2 = 0.80 from 6 hours post exposure; FiO2 = 0.40 from
Figure 4.5 Lung

morphology from pig lung 24 hours post phosgene exposure
(2000 mg min m−3) treated with protective ventilation. Little oedema or
inflammatory cell infiltrate was observed (A), with the majority of alve-
olar spaces (B) showing no evidence of pathology and an open struc-
ture comparable to air controls (*). Small accumulations of mucus and
cellular debris (↑) were observed in the lumens of a few of the conduct-
ing airways (C). Few focal areas of inflammatory cell infiltrate (*) were
observed in the submucosa of the conducting airways (D). Dark scales
are 200 µm and white scales are 50 µm. Stippled boxes show the area
expanded in the adjacent image.
6 hours post exposure; and FiO2 = 0.40 from 12 hours post exposure. Treat-
ment strategies continued as set until 24 hours post exposure.
The study demonstrated that increased inspired oxygen after a lethal dos-
age of phosgene significantly improved survival in all treated groups (24
hours). Arterial blood oxygenation was significantly improved even when
given at low flow (40% versus 80%) and delayed onset (started 12 hours post
exposure).88 The study suggests that a threshold concentration (∼40%) of
oxygen is required to improve survival and that delaying administration until
signs of exposure are apparent is not inferior to immediate therapy.
The benefits of protective ventilation, based on the ARDSNet protocol, and
supplemental inspired oxygen against phosgene injury have been demon-
strated.87,88 Use of protective ventilation and oxygen therapy is currently rec-
ommended to treat phosgene induced lung injury.24 However, Grainge and
Rice highlight the issues associated with electively intubating and ventilating
individuals following a small or large scale phosgene release. Local health-
care systems may not be able to deal effectively with large numbers of exposed
individuals requiring intensive supportive care due to the limited numbers
of ventilators and intensive care nurses. Surge capacity (the ability of health-
care systems to rapidly expand beyond normal services to meet the increased
demand in the event of large scale public health emergencies) needs to be
planned in advance. In order to spare scarce resources, oxygen therapy may
be delayed until the patient displays signs of poisoning, as determined by, for
example, reduced oxygen saturation.24
4.8 Recent Advances

As discussed above, traditional research approaches have relied on an under-
standing of the physiological responses and biological pathways affected
following exposure in animal models of phosgene induced lung injury. Dif-
ferentiating between the initiating events and downstream responses has
proved difficult and as such symptomatic therapeutic interventions, such as
administering corticosteroids as anti-inflammatories or β-agonists as bron-
chodilators, have proven largely unsuccessful. More recently, an increase in
our understanding of some of the downstream effectors following exposure
have led to a shift in our understanding of the potential mechanisms of phos-
gene injury.19 For example, innovations in genomics and proteomics tech-
nologies have resulted in the ability to identify changes in gene and protein
expression following exposure to toxic chemicals. By identifying the genes
that are up- or down-regulated it may be possible to identify potential thera-
peutic targets for future investigation (see Chapter 7 in Volume 2 for a more
detailed discussion). Genomics analysis allows assessment of the expression
of thousands of genes simultaneously in a single biological sample. Principal
component analysis (PCA) can then be used to reduce the complexity of the
data and simplify the task of identifying patterns and sources of variabil-
ity. Subsequently, advanced bioinformatics software, e.g. Ingenuity Pathway
Analysis (IPA, Ingenuity Systems, Inc., Redwood City, CA, USA), allows the
142 Chapter 4
most relevant signalling and metabolic pathways, molecular networks and
biological functions to be identified. Currently, the primary limitation of
genomics analysis lies in the fact that changes in protein expression, modifi-
cation or function can only be inferred.89 Proteomics analysis allows us to fill
this gap in our knowledge, using techniques such as liquid chromatography
combined with mass spectrometry to identify proteins in complex biologi-
cal mixtures. The issue of interpretation of changes in protein expression,
e.g. which are important in the injury mechanism and which are altered as a
result of downstream effects, continues to require consideration.
Sciuto et al. harvested lung tissue from mice exposed to a lethal challenge
of phosgene (32–42 mg m−3; 20 min) and used a genomics approach to iden-
tify the molecular mechanisms of phosgene injury that account for the phys-
iological and pathological disturbances known to be involved in phosgene
toxicity.61 These disturbances in lung tissue and bronchoalveolar lavage fluid
have been demonstrated in a range of animal species and include changes
to the GSH redox cycle, which is critical for the detoxification of free radical
mediated tissue and cellular injury.
The authors concluded that exposure to phosgene caused oxidative injury
in lung tissue over time, with the most significant changes in gene expres-
sion reflecting changes in GSH synthesis and redox regulation of the cell.
Glutamate–cysteine ligase catalytic subunit (GCLC; ϒ-glutamyl cysteine syn-
thetase), the rate limiting enzyme for GSH formation, was elevated as early
as 0.5 hours post exposure, suggesting that the lung is attempting to regulate
the antioxidant : oxidant ratio by increasing GSH synthesis soon after expo-
sure. Both glutathione peroxidase, important in the conversion of reduced
GSH into the oxidised form, GSSG, and glutathione reductase, which catal-
yses the reduction of GSSG back to GSH, were significantly increased from
4 to 12 hours post exposure. The changes reported support the hypothesis
that phosgene exposure increases the production of reactive oxygen species,
which cause changes in the redox status of the lung. In response, the genes
involved in regulation of the GSH redox cycle increase in an attempt to re-
establish normal antioxidant levels.
In contrast to the other antioxidant enzyme systems examined, there was
a decrease in the expression levels of superoxide dismutase (SOD3) that
persisted to 48 hours post exposure.61 This may indicate an overwhelming
formation of superoxide anions following phosgene exposure. The results
of this study are in agreement with previous observations by the authors.90
However, they are at odds with the studies by Jaskot et al., who reported an
increase in SOD activity from 1 to 3 days post phosgene exposure in rats
(2 mg m−3; 4 h).48 Differences in study design and choice of species may
explain the differences in response seen. It is likely, however, that antioxi-
dants such as GSH act protectively in the lung and bind to phosgene on
exposure, reducing GSH levels. When longer term exposures occur, the
endogenous reserves become overwhelmed and cellular damage occurs. The
lung’s response to the damage is to increase the synthesis and activation of
antioxidant enzymes, as demonstrated by Jaskot.
Liu et al., using gene expression profiles from lung tissue deposited in the
Gene Expression Omnibus (GEO) database by Sciuto et al., performed further
analysis of the molecular mechanisms of phosgene injury.91 They identified
582 differentially expressed genes in phosgene exposed mice compared with
air control animals. Mitochondrion organisation (critical to energy regulation)
was most significantly enriched, along with downregulation of transferase and
catalytic activity, suggesting changes in molecular function following phosgene
exposure. GSH metabolism was dysregulated, in agreement with the results of
Sciuto.61 Four genes in network clusters were also identified (F3, Meis1, Pvr
and Cdc6) that may have a role to play in phosgene induced lung injury.
Management of ALI from direct (e.g. phosgene inhalation) or indirect (e.g.
sepsis) causes remains a significant challenge clinically, where the same
severity of injury can have markedly different clinical outcomes, which are
difficult to predict. This variability in susceptibility to injury may be due to
the genetics of the individual concerned. Recently, Leikauf et al. sought to
investigate the role of genetics in an individual’s susceptibility to ALI.92
Forty-three inbred mouse strains were exposed to phosgene (4 mg m−3;
up to 24 hours) and survival times were recorded; there was a 4-fold differ-
ence in survival time between strains, with the most susceptible dying before
12 hours, and the more resistant strains surviving beyond 36 hours. Gross
pathology and histology indicated that pulmonary oedema, and the percent-
age of neutrophils and protein in the bronchoalveolar lavage fluid were more
evident in the sensitive strain (SM/J) than in the resistant strain (129X1/SvJ)
at early time points (6 and 12 hours).92
Transcriptomic analysis of lung mRNA from the sensitive mouse strain
(SM/J) identified a number of upregulated pathways compared with the insen-
sitive strain (129X1/SvJ), including apoptosis, unfolded protein response and
cytokine–cytokine receptor binding at 6 hours, along with GTPase regulator
activity and MAPK signalling at 12 hours. In addition, antioxidant response
transcripts mediated by the Nrf2 pathway [e.g. heme oxygenase (decycling)
1 (HMOX1) glutathione-S transferase 1 (GSTA1) and GCLC] were increased
more in the SM/J strain (at 6 hours only). Enriched pathways (increased tran-
scripts) in the resistant strain included lymphocyte activation, calcium ion
binding and T-cell receptor signalling. Common enriched pathways included
inflammatory response, cytokine activity and GSH metabolism (at 12 hours).
This study revealed 14 candidate genes with significant and 4 with sug-
gestive single nucleotide polymorphism (SNP) associations, some of which
could be associated with ALI (Table 4.4). Understanding the changes in gene
expression may lead to the identification of novel therapeutic pathways for
investigation, such as the use of inhibitors for Alox5 to reduce ALI.92,93
4.8.1 Potential Future Therapeutic Options

The lung is the largest epithelial surface in the human body, the alveolar
surface area being estimated at 70 m2. It is highly complex and constitutes
a three dimensional structure of unique cells lining a series of bifurcating
144 Chapter 4
Table 4.4 Potential role of some candidate genes in acute lung injury. a
Candidate genes with

significant SNP associations Potential role in acute lung injury
Atp1a1 Maintains electrochemical gradient of Na+/K+ ions
across plasma membranes; essential for osmoreg-
ulation; role in clearance of pulmonary oedema
Alox5 Encodes arachidonate-5-lipoxygenase enzyme,
which converts arachidonic acid to leukotrienes—
important mediators of a number of inflamma-
tory and allergic conditions
Hrh1 Gene encoding histamine receptor H1. Histamine
mediates contraction of smooth muscle and an
increase in capillary permeability due to contrac-
tion of terminal venules
Plxnd1 Plexin D1 expressed in pulmonary vasculature—sig-
nificantly associated with phosgene survival
Ptprt Member of the protein tyrosine phosphatase (PTP)
family—regulates a variety of cellular processes
including cell growth, differentiation, mitotic
cycle and oncogenic transformation
Reln Gene encodes a large secreted extracellular matrix
protein thought to control cell–cell interactions
critical for cell positioning and neuronal migra-
tion during brain development
Candidate genes with suggestive SNP associations
Itga9 The protein encoded by this gene forms an integrin
that is a receptor for VCAM1. Also associated with
TGF-β1 signalling in the lung
Mapk14 The protein encoded by this gene is a member of the
MAPK family and is involved in a wide variety of
cellular processes such as proliferation, differenti-
ation, transcription regulation and development
Vwf Von Willebrand factor is important in the mainte-
nance of haemostasis, promoting adhesion of
platelets to sites of vascular injury
a
Table complied from Leikauf et al.92
tubes. There are reported to be more than 40 different cell types in the adult
human lung, many of which are involved in the maintenance of lung struc-
ture and function, as well as repair processes. However, while cells such as
the Clara, goblet, ciliated and basal cells are restricted to the airways distal
to the alveoli, only the cuboidal type 2 pneumocytes and squamous type 1
pneumocytes (constituting ∼95% of the alveolus) cover the lining of the alve-
olus itself. The type 2 cell is regarded as the local progenitor cell for the epi-
thelial surface of the alveolus, as well as the cell responsible for synthesising
and secreting surface active pulmonary surfactant.28,81 Damage to type 1 cells
can have a profound effect on gas exchange, especially if the damage results
in accumulation of pulmonary oedema within the alveolus and surfactant
function is impaired. Exposure to lung damaging chemicals such as phos-
gene causes damage to the epithelial–endothelial barrier of the alveolus,
resulting in an early influx of proteinaceous oedema into the lumen of the
alveoli, which is postulated to compromise surfactant function.28 Re-estab-
lishment of this blood–air barrier is a critical requirement in the treatment of
the ALI that results. A major complicating factor to this repair process, how-
ever, is that the alveolar and airway epithelium have a low capacity for regen-
eration and an intrinsically lower level of cellular turnover when compared
with other epithelial organs, e.g. the skin. The potential use of stem cells and
growth factors in the treatment of ALI/ARDS, which may have potential use in
the management of phosgene induced ALI, are discussed in this section.94,95
4.8.1.1 Stem Cells
Angelini et al. reviewed the potential options for stem cell therapy in the treat-
ment of ALI/ARDS from direct (e.g. toxic inhalation injury) and indirect (e.g.
sepsis) damage to the lungs.94 The authors provide an overview of the differ-
ent stem cell types (resident lung stem cells, bone marrow derived stem cells,
mesenchymal stem cells, embryonic stem cells and induced pluripotent stem
cells) and discuss their potential in pulmonary injury therapy.94 Phosgene is
known to cause ALI, which can progress to an ARDS, via a direct effect on the
lungs, and the authors suggest that stem cells, either as a single or combina-
tion of populations, may present a unique opportunity as a therapeutic option
for chemically induced ALI. However, this approach is currently some way from
being available to the clinician, as the availability of the stem cells themselves
and the techniques required to scale up production continue to be developed,
and clinical trials in human patients are performed.
4.8.1.2 Growth Factors
Lindsay provides an insight into the regulation of repair mechanisms in
the lung following ALI/ARDS from different aetiologies (direct and indi-
rect).95 The author provides a clear indication of the roles of the various cells
involved in lung injury and repair, and of the role of growth factors in these
processes. Although many promising candidate pharmacological treatments
have been evaluated for the treatment of chemically induced lung injury,
their clinical value is often debatable. As such, despite improvements in
ventilation strategies such as ARDSNet protective ventilation, pharmaco-
logical intervention for ALI/ARDS remains problematical. A new approach
is required for the treatment of severely compromised lungs. Therapeutic
interventions directed at upregulating the repair of the damaged alveolar–
capillary blood–air barrier may be of value, particularly with respect to chem-
ically induced injury. Lindsay reports that keratinocyte growth factor (KGF),
epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) are
emerging as the most important candidates. While hepatocyte growth fac-
tor (HGF) does not have epithelial specificity for lung tissue, the enhanced
effects of combinations of growth factors, such as the synergistic effect of
146 Chapter 4
HGF on vascular endothelial growth factor (VEGF) mediated endothelial cell
activity, and the combined effect of HGF and KGF in tissue repair should be
investigated, particularly as the latter pair of growth factors are frequently
implicated in processes associated with the repair of lung damage. Synergis-
tic interactions are also reported to occur between trefoil factor family (TFF)
peptides and growth factors such as EGF. TFF peptides may provide value
as a short term therapeutic intervention in stimulating epithelial spreading
activities and allowing damaged mucosal surfaces to be rapidly covered by
epithelial cells, essential for the repair of barrier function.
Beneficial effects of growth factors such as KGF have been shown in some
animal models of ALI, through the improvement of endothelial and epithe-
lial barrier function, and enhanced rate of alveolar fluid clearance. Further
animal studies and clinical trials are required to confirm this approach. The
effect of KGF on pulmonary dysfunction in ALI patients is under investigation
in a randomised placebo-controlled trial (KARE).96 This trial will assess the
benefit of palifermin (recombinant KGF) in ALI patients. The use of growth
factors against chemically induced lung injury may well be influenced by the
outcome of such clinical trials in ALI patients.
4.9 Conclusions
Review of the literature has identified many studies investigating the mech-
anisms of action of phosgene induced lung injury, as well as therapeutic
options. The studies involve numerous in vitro and in vivo models of phosgene
poisoning and most agree that phosgene’s toxic effects are by direct action in
the lower respiratory tract where damage to the alveolar–capillary blood–air
barrier occurs. This results in the accumulation of proteinaceous pulmonary
oedema fluid in the alveolar regions as well as an accumulation of inflamma-
tory cells. When activated, these cells produce further damage through release
of reactive oxygen species. Many studies have identified the importance of oxi-
dant systems, especially those involving GSH, in the development of phosgene
induced lung injury. Damage to the surfactant system results in the lungs no
longer being able to efficiently provide oxygen to the tissues and death from
hypoxia may result in severe cases. However, our understanding of the initi-
ating events that result in this injury are not complete; in particular, events
occurring during the non-symptomatic latency period have not been fully
resolved. Future studies should focus on these early initiating events to clearly
define cellular interactions that lead to downstream signalling pathways being
activated. New techniques, such as more complex in vitro systems or transcrip-
tomics approaches, continue to evolve and should improve our understanding
of these mechanisms and therefore offer the potential to provide information
on new pathways, including those at the cellular level, that may be targeted
for therapeutic intervention. Studies that focus on limiting the severity of
injury as well as the ability to enhance the capacity of the lung to recover (e.g.
through the use of growth factors that can stimulate epithelial repair) should
also be performed. These studies may provide clinical options beyond the use
of supportive care, which is likely to be overwhelmed should large numbers
of casualties occur following accidental or deliberate release of phosgene. In
these situations, the lack of diagnostic indicators of injury is likely to result in
delays in the treatment of patients, most of whom will be non-symptomatic for
some hours after exposure has occurred.
Far from being confined to use in warfare, phosgene remains an important
global TIC used in the production of numerous industrial and pharmaceutical
products, and as such, despite restrictions on its storage and supply, exposure
remains a potential problem. Despite decades of research into its mechanisms
of action, and recent increases in our understanding of the biochemical and
physiological pathways involved, evidence based treatment guidelines are
lacking, with supportive care (ventilation and oxygenation) remaining the
mainstay for treating the resulting injury. Advances in our understanding of
the limitations of animal models and extrapolation of the effects to man will
continue to provide new options for therapeutic approaches.
Acknowledgements
The author wishes to thank to Dr Chris Green, Dr John Tattersall and Dr John
Jenner (Dstl) for their very useful contributions and support throughout the
writing of this chapter, and Mr Chris Taylor (Dstl) for the production of his-
tology micrographs. The author would also like to acknowledge Dr Chris
Grainge and Dr Emily Swindle (Faculty of Medicine, University of Southamp-
ton) for their technical expertise in the preparation of the human bronchial
epithelial cell air–liquid interface model.
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Chapter 5
Human Exposures to Sulfur

Mustard
John Jenner*a
a
Toxicology, Trauma and Medicine Group, CBR Division,
Dstl, Porton Down, Salisbury SP4 0JQ, UK
*E-mail: jjenner@dstl.gov.uk
5.1 Introduction
Sulfur mustard (SM) is unique among chemical warfare agents because
of the large number of reports of its effects in man. The majority of these
reports are of its effects after release on the battle field, and give a descrip-
tion of the types of effect and their time course from exposure to resolution
of the injury.1,2 However, SM is also one of the few chemicals that have been
the subject of tests on humans to determine how toxic they are in terms of
the doses or dosages that produce toxic effects. Unlike reports of acciden-
tal or battlefield exposures, these trials were carried out in chambers under
controlled, or at least carefully recorded, conditions, usually with analytical
confirmation of chamber concentrations. Many of the reports of these trials,
which were classified at the time they were produced, have now been released
into the public record and are available for scientific review. This chapter
reviews those reports that are now available to the general public in addition
to the work already published. Volunteer trials were carried out in the USA,
UK, India and Australia. The reports of these trials that have been released
to the public record are held by the Defense Technical Information Service

154
Human Exposures to Sulfur Mustard 155
in the USA (http://www.dtic.mil), the National Archives in the UK (http://
www.nationalarchives.gov.uk) and Australia (http://www.naa.gov.au). All of
the defence reports quoted in this chapter are available from the websites of
these record offices.
Although these trials would be considered unethical today, the reports of
them remain an invaluable source of toxicological information and their use
in scientific research should be regarded as a tribute to the sacrifices made
by those who took part.
Most volunteer trials to determine the effects of chemical warfare agents
were carried out on three agents, SM, sarin (GB) and VX. Widely used in the
First World War (WWI), SM was the subject of many trials between 1919 and
1945, whilst the discovery of large stockpiles of GB in Germany in 1945 led
the western world to investigate this agent extensively in humans in the late
1940s and 1950s. Some of the trials with GB are reviewed in Chapter 3. The
series of trials with SM carried out in Australia in WWII have been extensively
described in the open literature,3,4 but at the time of writing, the original
research reports of these trials remain embargoed by the Australian National
Archive whilst their release status is reviewed.
5.2 Toxic Effects of SM in Humans

The toxic effects of SM in humans and animals are described in detail else-
where,2,5,6 but as an introduction to what follows in this chapter it is import-
ant to realise that SM, as vapour or liquid, enters the body through virtually
every surface it comes into contact with. SM is not an immediate irritant so
there are no obvious signs or symptoms during exposure, but after a latency
of a few hours (6–12 hours dependent on dosage) the surfaces exposed
become inflamed. This inflammation can become so severe that the skin
blisters, eyelids go into spasm and lung function is seriously impaired due to
sloughing of the lining of the major airways.7,8 SM injuries heal very slowly,
probably because of the damage to surrounding tissues caused by alkylation
of cellular constituents.9,10
It was clear from early observations,11,12 confirmed during more recent
conflicts,5,13 that dosages normally received during battlefield exposure were
not usually sufficient to kill, but produced some very serious incapacitating
injuries. Such injuries are very slow to heal and produce a variety of long term
effects on health, such as cancer,14–18 recurrent skin lesions,19,20 keratosis of
the eye21 and respiratory difficulties.22 The most obvious injuries caused to
the skin resemble thermal injury and are often described, perhaps mislead-
ingly, as burns. These effects are described in more detail below.
5.2.1 Effects on the Eyes

The effects of SM on human eyes were extensively described in casualties
from WWI. Although the original reports of these casualties have not been
located, they are described in the scientific literature of the time. Warthin23
156 Chapter 5
describes in detail the effects that SM has upon the eyes from French and
Italian sources.24–26 These early reports speak of conjunctivitis occurring
within 6–12 hours of exposure. In general, the effects described are restricted
to mild to severe conjunctivitis, resulting in some cases in a reflex closure
of the eyes (blepharospasm), photophobia and lacrimation that, although
incapacitating at the time, resolved within a few days to a week. Infection was
absent in mild cases. The more severe cases, involving panophthalmitis and
ulceration of the cornea, were few. Teulieres24,25 describes some 1500 cases
of eye injury, of which only four presented with these more severe symptoms.
The more severe cases were resistant to treatment, slow to heal and produced
some permanent impairment of sight.
A large number of injuries occurred in the production of SM between the
World Wars. A summary of all injuries in the UK showed that 10.4% of the
939 eye casualties showed improvement of corneal injuries with time and
only 1% had severe corneal lesions;27 similar casualty rates for liquid and
vapour exposure have been reported from the USA.28 Unfortunately, there
were no long term follow-up studies of these patients to determine their ocu-
lar status years after exposure.
The British reported many thousands of eye casualties during WWI and
SM was responsible for 77% of these. Analysis of the total figures reveals that
75% of cases were relatively mild, amounting to conjunctival irritation requir-
ing an average stay in hospital of 2 weeks.29,30 Another 15% were described
as moderate, with incapacitation for 4–6 weeks. Finally, 10% were described
as severe requiring periods of up to 6 months in hospital for symptoms to
stabilise.30 A total of 51 British soldiers were reported as blinded and there
were 180 vision related pensions.31
Acute severe injury of the eye with SM might result in recurrent corneal
ulcerative disease for the remainder of the patient’s life, with a maximum
incidence occurring 15–20 years after the initial injury.21,32 Based on exten-
sive data, there is a causal relationship between severe exposure to SM and
the development of delayed recurrent keratitis.
Warthin23 compared clinical descriptions with the pathology of experi-
mentally induced lesions in the eyes of rabbits and concluded that the inju-
ries were very similar. The injuries produced by dilute liquid contamination
of the eye and by vapour exposure were essentially identical in rabbits and
humans. The injuries were dose dependent, with higher concentrations and/
or longer exposure times producing more severe injuries. The latency period
before symptoms were observed was also dose dependent, with higher con-
centrations producing symptoms more rapidly. Injury to the cornea is critical
to loss of sight or permanent injury. Five “well recognised stages” of severe
corneal damage are described by Hughes:30

1 Immediate damage to the corneal epithelium with oedematous cloud-
ing and necrosis of the stroma;
2 After 5 hours, an infiltration of polymorphonuclear cells at the schlero-
corneal junction, thence extending into the corneal stroma;
3 At 5–7 days there is a clinical improvement of the opacity with dimin-
ished oedema of the stroma;
4 A progressive vascularisation of the cornea extending in from the limbal
vessels that may continue for several weeks;
5 Persistent ulceration of the cornea for weeks, or recurrent ulceration
after a latent period of years.

The long term effects of SM poisoning have been described in casualties
surviving gassing during WWI. Mann21 described 84 cases of delayed ker-
atitis in WWI gas victims with corneal degeneration, varicose conjunctival
and corneal vessels, and vascular scars on the interpalpebral conjunctiva.
The majority of cases of keratitis described by Mann21 were delayed in onset
by between 10 and 20 years, and were not found in patients whose initial
symptoms lasted less than 8 weeks. Patients did not generally associate the
keratitis with gassing. The visual acuity in all but 2 of the 84 cases reviewed
by Mann was improved by the use of contact lenses.
Although invaluable in describing the effects of SM on the eyes,
reports from gassings during WWI were not associated with any definite
measure of the exposure concentration or duration. Work to define the
dosage that would produce injury to the eyes was reported soon after the
end of WWI. Reed32 reported trials on 13 human volunteers to determine
the concentration that would produce effects on the eyes. One eye was
protected as a control and the other exposed to SM vapour (0.47–0.58 mg
m−3) for periods of time between 10 and 25 min (Table 5.1). The results
were recorded as the numbers of exposed men developing conjunctivitis
and, although variable, support the conclusion that concentration–time
products (Cts) above 5 mg min m−3 may produce conjunctivitis. However,
an inherent assumption allowing this conclusion was that Ct was con-
stant (i.e. Haber’s law applied), which at that time was not considered
or confirmed, although future studies indicated that this was true (see
below).
Two studies performed during the Second World War (WWII) further
defined the relationship between the effects of SM on human eyes and the
dosage of vapour in terms of exposure concentration (C) and duration of expo-
sure (t). Guild and Harrison33 exposed volunteers in temperate conditions
Table 5.1 Incidence of conjunctivitis in men exposed to SM vapour.32

Analytical concentration Duration of Ct (mg min Number Number with
(mg m−3) exposure (min) m−3) exposed conjunctivitis
0.48 10 4.8 2 2
0.47 20 9.4 3 1
0.48 25 12 2 1
0.58 10 5.8 2 2
0.55 20 11 2 0
0.58 20 11.6 2 2
158 Chapter 5
(although temperature and humidity were not specified) and Andersen34
performed exposures under hot weather conditions on the Indian subcon-
tinent. Both authors described similar dose related symptoms of exposure
progressing from minor bulbar injection at low concentrations or exposure
times through to more generalised conjunctivitis to a severe conjunctivi-
tis, photophobia and blepharospasm. Both studies concluded that over the
range of exposure times investigated (15 seconds to 24 hours) the effects
were directly related to the Ct (i.e. Haber’s law applied). Guild and Harrison
describe the results of investigations of a wide range of concentrations and
exposure times, while the range of exposure times utilised by Anderson was
not as large.
Although it is always difficult to analyse the subjective description of clin-
ical signs, in this case the effects reported by both Guild and Harrison, and
Anderson can be categorised as:

●● “Minor effect”: slight injection and minor irritation of the eyes
●● “Definite effect”: generalised injection and conjunctivitis
●● “Casualty”: severe conjunctivitis, possibly with photophobia and
blepharospasm

Using this category system, the results of these two studies can be ana-
lysed to derive a toxic load exponent (TLE; Figure 5.1). For each effect the
TLE is close to 1 over the concentration range investigated indicating that
Haber’s law applies to the effects of SM on the eyes. The authors concluded
that under temperate conditions, the range of Cts where incapacitating
conjunctivitis, photophobia and blepharospasm occurred was 70–100 mg
min m−3, but under hot wet conditions this range was reduced to 60–90 mg
min m−3. Since all of the exposures in both studies produced some effect
on the eyes, a “no observable adverse effect level” was not established, but
the lowest Ct used in either study was 12.5 mg min m−3. These observations
are consistent with the Cts producing the effects described in Figure 5.1,
but these Cts are the ECt100 (dose to produce 100% decrease of pupil area)
since the data points all represent individuals who have shown the effect.
The ECt50 values for these effects would be lower and are not derivable
from the data in the papers because of the low numbers exposed at each
exposure time.
5.2.2 Effects on the Skin

Contact with either vapour or liquid SM produces a characteristic effect on
human skin that has previously been described in many publications35,36 and
in Chapter 2 of this Volume and Chapter 4 of Volume 2. Briefly, after a latency
period that is longer at lower doses, the first effects observed are those asso-
ciated with inflammation. The skin goes red (erythema) due to vasodilation
in the affected area. At low doses this may be the only effect or may be fol-
lowed by dry desquamation of the area some days later. At higher doses the
Figure 5.1 Concentration–exposure

time relationship for SM effects on the eyes.
Plots of pooled data from human exposures carried out in the UK33
and India34 based on the model: log t = m log EC + log K where t is expo-
sure time, EC is the effective concentration, K is a constant unique to
the toxic effect and m is the toxic load exponent. The effects are those
described by the authors as “minor effect” (fine bulbar injection), “defi-
nite effect” (conjunctivitis) and as “casualty or just short of casualty”
(not well defined in the source documents but probably severe conjunc-
tivitis or blepharospasm producing temporary blindness). The simi-
larity of the EC values for conjunctivitis and “definite effect” probably
reflects the poor fit to the data for the former as demonstrated by the
lower r2 value for that regression line.
160 Chapter 5
skin becomes oedematous as fluid diffuses into the tissue from the blood
and some micro-vesicles may form. At higher doses still, the micro-vesicles
coalesce into large blisters or bullae that are filled with cloudy clear or yel-
lowish fluid. After some time, usually days, the blisters will burst and the
area of skin becomes denuded of surface tissue. An eschar of dead skin tissue
that impedes re-epithelialisation may form and is usually removed by some
form of debridement to speed healing. Some or all of the pathologies have
been observed in most of the volunteers in the trials described below, whether
exposed to vapour or to liquid. To date, no animal has been identified that
produces coalesced blisters in quite the same way as humans. Hairless
guinea pigs37 and pigs have been reported to produce micro-vesicles, but the
large, pendulous, fluid filled blisters appear to be unique to humans. Aside
from the blisters, however, the injury produced in animal skin by SM is very
similar to that produced in humans: an initial inflammatory response fol-
lowed by tissue destruction and the formation of an eschar that compromises
healing.
5.2.2.1 Vapour
Human volunteers have been exposed to SM vapour, either whole body or on
selected regions of the body, normally the arm. In addition, whole body expo-
sures have been undertaken in a controlled chamber environment and in the
field, under a variety of climatic conditions. Exposures in chambers are more
accurate, because, although the field trials may have used vapour capture
devices to sample the air, the variation in vapour concentration during the
exposure could not be accounted for.
5.2.2.1.1 Exposure of the Forearm. Several groups have attempted to estab-

lish the threshold Ct for erythema in humans. The first systematic studies
were probably those reported by Marshall et al.38 This group investigated dif-
ferences in susceptibility of individuals to mustard gas contamination of the
skin by measuring the minimum amount of time to cause erythema on fore-
arm skin exposed to saturated vapour at 20 °C. Vapour was generated using
a cotton plug soaked in liquid SM, in a small test tube inside a larger tube
containing water, both were then placed in a water bath at 20 °C. Prepared
tubes were left for a time to allow any other chemicals that may have been in
the tubes to evaporate before the addition of SM. Exposures were carried out
by holding the mouth of the small tube firmly on the forearm for a fixed time.
There was a marked variation in the amount of time it took to produce a
burn with saturated vapour (n = 54) and “coloured” skin seemed to be less sus-
ceptible than white skin. Reaction times of between 1 and 600 seconds were
recorded and their distribution approximates to a log-normal distribution.
These figures must be interpreted with some caution. The authors com-
ment on differences in the vesicating potency of different “batches” of SM,
probably due to differences in the purity of the agent, which was not analyt-
ically confirmed.
39
The results reported by Nagy et al. appear consistent with the work of
Marshall et al. Using a vapour generating device, Nagy and co-workers mea-
sured the amount of SM penetrating the skin of the forearms of human vol-
unteers by measuring the SM remaining in the applicator at various times
after application and related this to the degree of injury at 48 hours. Cts of
2300–7700 mg min m−3 (temperature = 21–23 °C; t = 3–10 min) and 2900–
8800 mg min m−3 (temperature = 30.6 °C; t = 2–6 min) all produced erythema
or vesication on the forearms of 6–12 volunteers. However, this report does
not describe the time course of the lesion and the reader is left to assume
that the 48 hour lesion is the most severe reaction.
5.2.2.1.2 Whole Body Exposures
Chamber Trials. In a series of chamber exposures carried out in India in

1942 40, men were exposed to Cts of SM from 40.9 to 176 mg min m−3 (C = 1.8–
26 mg m−3; t = 5–63 min). In the first series of exposures men were exposed in
groups of three or four wearing light anti-gas suits and respirators. Holes, 2″
in diameter were cut into the arms and legs of the suits with the right hand
exposed in order to establish the Ct for the next series of experiments. In the
next series, men were exposed wearing oilskin trousers and open neck cot-
ton shirts with the sleeves rolled up (plus respirators and rubber boots). In
the third series of experiments, the men wore ordinary khaki drill and open
necked shirts but oilskin underpants to protect the genital region. In the
final series of experiments the men wore ordinary tropical battle dress. The
exposures are summarised in Table 5.2.
None of those in exposure 1 (40.9 mg min m−3) showed a reaction, the most
severe reaction in exposures 2 and 3 (70 and 68 mg min m−3) was a raised
erythema and some pigmentation, whilst exposure 4 (116.6 mg min m−3) pro-
duced nothing more than a trace erythema of the hand.
The exposure that formed series two (137 mg min m−3) produced an ery-
thema of the chest, back and shoulders in one of the subjects but nothing
more than a trace erythema of the upper arms and chest in the other two.
Exposure 8 (series 3; 144 mg min m−3) produced a generalised low grade
erythema over the chest, back, arms and legs of all three subjects with the
areas protected by the respirator and protective shorts delineated in some.
Some of the areas showed pigmented erythema in one subject.
The exposures of unprotected men carried out in series 4 produced more
marked effects. Exposure 9 (69 mg min m−3) produced irritable crotch and
scrotal erythema in one of the three subjects, itching of the knee flexures in
one of the three, and trace erythema of the chest, back and arms or thighs
in two of the three. Similar reactions were produced by exposure 10 (110
mg min m−3) except that one subject developed a “papular erythema” of the
chest and back. No reaction in the crotch is mentioned in the other two.
Exposure 11 (113.4 mg min m−3) produced few visible effects with trace ery-
thema on the back and scrotum of one subject and complaints of irritation
in the other two. Exposure 12 (162 mg min m−3) produced a generalised
162 Chapter 5
Table 5.2 Summary
of exposures to SM vapour carried out at Rawalpindi, India
(1942).40
Mean Number
Exposure Date temperature Minimum C (mg Ct (mg of
number (D/M/YY) (°F) RH (%) m−3) t (min) min m−3) subjects
SERIES 1
1 23/7/42 89 49 6.25 7.5 40.9 4
2 27/7/42 91 51 7 10 70 4
3 29/7/42 95 44 6.8 10 68 2
4 31/7/42 89 51 10.6 11 116.6 3
5 18/8/42 80 56 9 15 135 4
6 19/8/42 84 62 6 26 156 4
SERIES 2
7 2/9/42 86 51 10.3 13.3 137 3
SERIES 3
8 3/9/42 85 64 12 12 144 3
SERIES 4
9 7/9/42 86 57 13.8 5 69 3
10 8/9/42 86 50 20 5.5 110 3
11 16/9/42 92 39 1.8 63 113.4 3
12 17/9/42 92 42 2.7 60 162 3
13 18/9/42 86 46 5 25 125 3
14 24/9/42 73 57 3.2 35 112 3
15 28/9/42 80 31 8.85 15 132 4
16 30/9/42 80 19 8.4 21 176 3
erythema with vesication of the arms and torso of some subjects. The gen-
italia were erythematous and desquamated in two of the three and were
irritated in the third. The injuries were considered of casualty severity in
all three subjects. Exposure 13 (125 mg min m−3) produced similar but less
severe injuries. A widespread erythema involved the arms, neck, chest,
back and flanks, and the flexures of the limbs were irritated. In two of the
three the scrotum was irritated and in one case desquamated. The clothing
was worn for 4 hours after exposure 14 (112 mg min m−3) with no more
effect than a mild irritation of the torso, and an irritable crotch in two of
the three subjects. At the end of the last two exposures (15 and 16) the
subjects are recorded as leaving the chamber with cool dry skin. Exposure
15 (132 mg min m−3) produced mild to moderate erythema and irritation
of the axillae and scrotum in the two subjects who wore their clothes for
4 hours after exposure. The two subjects who bathed and changed imme-
diately after exposure experienced very little effect. Exposure 16 (176 mg
min m−3) produced similar results with the two subjects who bathed and
changed immediately after exposure experiencing little effect other than an
irritable crotch. One subject who remained in his clothes for 4 hours after
exposure developed erythema of the back and irritation of the scrotum with
dry desquamation.
Two subjects were exposed to a Ct of 750 mg min m−3 for 16 min [87 °F
and 84% relative humidity (RH)].43 These men were protected with CC-2
(N,N′-dichloro-bis[2,4,6-trichlorophenyl] urea) impregnated drawers but
were badly burned over the rest of their bodies. They were hospitalised for
19 and 28 days.
In a series of trials carried out between 1944 and 1945 Heinen and co-work-
ers41 exposed a total of 212 men to SM in 33 separate tests. Men were exposed
in a purpose built chamber42 to Cts of 54–695 mg min m−3 (C = 1.6–12 mg m−3,
t = 30–60 min, RH = 35–86%). Men were dressed in skivvy shirts, Nainsbrook
shorts, watch caps, blue denim shirts, dungaree pants, standard socks and
shoes. In some tests the Nainsbrook shorts were replaced by CC-2 impreg-
nated shorts of the rib-knit variety impregnated by the aqueous process (0.5
mg Cl cm−2). In others, carbon coated cloth suspenders were worn. No men-
tion is made in the report of the length of time the men wore their clothes
after exposure. The men did not exercise during exposure and led sedentary
lives before and after exposure with occasional mild athletics.
Heinen et al. made the general observation that the mild erythema he recorded
was not a good measure of a threshold effect because it could be easily confused
with erythema resulting from causes other than the SM challenge. Moreover,
none of the men exposed during these tests showed “actual bleb formation”,
which was observed in the Australian studies. Under similar conditions of RH
and temperature there was a dose dependent increase in severity and extent of
the lesions produced by Cts of between 50 and 600 mg min m−3 (Figure 5.2).
5.2.2.1.3 Quantification of Skin Burns. There have been a number of

attempts to devise methods of quantifying skin burns in terms of severity
and extent. The two most useful appear to be that developed at Porton and
used by Sinclair to report on his own studies and on the clinical aspects of
the Australian trials, and that used by Heinen et al.41 Both systems are essen-
tially subjective scoring systems based upon mild, moderate and severe ery-
thema and oedema, desquamation and frank vesication. In order to make a
more informed assessment of the data from the chamber trials carried out
in India and the USA, the results have been converted to the scoring system
proposed and used by Heinen et al. and are summarised in Figure 5.2.
5.2.2.2 Effects of Increased Temperature on SM Injury

The basic tests of SM vapour carried out by Heinen et al.41 revealed that a severe
generalised erythema was achieved by exposure to a Ct of 200 mg min m−3 at
100 °F (38 °C) or 200–300 mg min m−3 at 90 °F (32 °C), but at a temperature of
70 °F (21 °C) this reaction was not achieved even by a Ct of 500–600 mg min m−3.
However, a Ct of 600 mg min m−3 produced such severe reactions in the genitalia
and the axillae at low temperatures (60–70 °F, 16–21 °C) that the sensitivity of the
general body surface at low temperatures could not be determined.
There are no reports available to the author to quantify the effect of increas-
ing temperature alone on the Ct of SM required to produce effects. How-
ever, all of the studies that have compared the effects of SM in a variety of
164 Chapter 5
Figure 5.2 Graphical

representation of the dose–effect relationship of SM. Bars
represent graded reactions of different areas of the skin to SM vapour
in humans. Reactions are compiled from US41 and CDRE (India)
reports40,43 for ranges of dosages specified. Severity: 1: mild erythema;
2: moderate erythema; 3: intense erythema; 4: erythema with oedema,
maceration of axillary skin and dry desquamation; 5: vesicle or numer-
ous micro-vesicles or crusting and ulceration of the scrotum or axillae.
Body areas: abd: abdomen; arm: arm; ax: axillae; bt: buttocks; cf: cubi-
tal fossae; dth: dorsal thorax; ing: inguinal region; leg: leg; lth: lateral
thorax; neck: neck; pen: penis; pop: popliteal fossae; scr: scrotum; sh:
shoulder; thi: thigh; vth: ventral thorax; wrt: wrist.
conditions have shown that effects are produced at lower Cts on hot wet skin
than on cool dry skin. Renshaw44 concluded that this relationship is due to a
layer of water on the skin. Although this study was not well controlled, Ren-
shaw concluded, from a limited number of exposures, that a layer of water
increased the vesicating potential of SM. The presence of NaCl in the water,
or a filter paper on the surface of the skin made no difference, provided a
continuous layer of water was present. As with the experiments by Nagy et
al.,39 this was based on the lesions measured at 48 hours after challenge and
no descriptions of lesion progression are given.
5.2.2.3 Effects of RH on SM Injury
There does appear to be an increase in the severity and extent of the resulting
lesion when the RH is increased at a constant temperature, but the data are
less convincing than those for a dependence on temperature. Increasing the
temperature seemed to decrease this effect. There was some indication that
the conditions outside the exposure chamber also affected the level of injury
produced. Exposures carried out in spring and summer under the same con-
ditions of temperature and humidity in the exposure chamber appeared to
show that above a Ct of 150 mg min m−3 there was an appreciable increase
in the severity of the skin damage.41 However, the number of exposures car-
ried out and the variation in responses seen across the study confound any
clear conclusion. It is not clear in these studies if the men changed their
clothing soon after exposure. If, as was common practice in other studies,
they remained in the clothes that they were exposed in for several hours after
exposure the possibility that they continued to absorb vapour from their
clothes cannot be excluded. The ambient temperature and humidity would
influence such post exposure absorption.
The observation of Heinen et al.41 that injuries to the penis and scrotum
produce casualties and that the neck is a sensitive area, is consistent with
other studies.
In a series of “special tests” Heinen et al.41 investigated the effect of sweat-
ing, drying the skin and lanolin on the response to SM vapour. The results
were consistent with the hypothesis that the degree of sweating determines
the severity and extent of the lesions. Cooling of the subjects prior to expo-
sure reduced the severity of the injury, as did drying the skin with aluminium
chloride powder, which was graphically demonstrated by drying one side of
the scrotum in some subjects.
Heinen et al.41 concluded that whilst temperature did not change the sever-
ity of the burns of the genitalia and axillae, increasing the temperature did
change the configuration of the axilla injury from a central lesion at low tem-
peratures to a more generalised injury that spared the central area at 90 °F
(32 °C) and above. There was a generalised intense erythema produced by
a Ct of 250 mg min m−3 at 90 °F and by 200 mg min m−3 at 100 °F (37.7 °C).
Moreover, a Ct of 500 mg min m−3 at 70 °F (21 °C) and 600 mg min m−3 at 60
°F (16 °C) only produced moderate erythema over most of the body but more
severe lesions in the genitalia and axillae.
5.2.2.4 Analytical Considerations
Modern methods of experimentation would require the purity of the SM
to be specified and the concentrations in chambers to be analytically con-
firmed. In most of the chamber trials carried out in WWII these criteria
were fulfilled, although the methods used were not always described, or
referenced, in each report. However, good details of the concentrations
achieved in the chambers are given within the individual experimental
reports from the USA. In addition, the performance of the analytical meth-
ods in common use by each nation at the time that these studies were con-
ducted are summarised in Porton Memorandum 19 45 and NRLR P-2208.46
The two methods that were in routine use were the iodoplatinate method
and the bromine method. The performance of these methods in detecting
166 Chapter 5
SM and its breakdown products are summarised in Porton report 2377 47
and Porton memorandum 19.45
5.2.2.5 Clothing
Some authors have concluded that wearing ordinary clothes does not affect
the development of SM injury; however, the effect of removing clothing
immediately after exposure suggests that this is probably not a valid conclu-
sion. The length of time for which the clothes were worn after exposure may
be important, as shown by the trials at Rawalpindi40 where the removal of
clothing and bathing immediately after exposure eliminated the injury sus-
tained by subjects exposed concurrently who continued to wear their cloth-
ing for 4 hours after exposure. This is probably due to the SM absorbed by
the clothing continuing to be delivered to the skin after the exposure was
complete. In hot summer weather the rate of evaporation and penetration of
clothing by SM would be higher. It is also known that highly hydrated skin is
more easily penetrated by chemicals generally, so the presence of sweat on
the surface of the skin and higher water content in the stratum corneum as
part of physiological thermoregulation would increase the dose of SM pene-
trating the skin at the same challenge concentration.
Moreover, most reports highlight injuries to the neck and wrists, which
are described as more sensitive to the effects of SM than other areas. How-
ever, pictures presented by Heinen et al.41 delineate the neck-line of the cloth-
ing, consistent with the clothes worn protecting the skin of the shoulders
and torso but not the neck. The clothing status of volunteers in the studies
quoted above are summarised in Table 5.3.
Table 5.3 Clothing

worn during chamber exposures to SM.
Duration of wear after
Report Clothing worn during exposure exposure
Heinen et al., Skivvy shirts, Nainsbrook shorts, Not specified
1945 41 watch caps, blue denim shirts, dun-
garee pants, standard socks and
shoes, and service respirators
CDRE India Series 1: light anti-gas suits and respi- 4 hours except one
report 245 40 rators, holes cut into arms and legs exposure where two
of the suits, right hand exposed subjects changed and
Series 2: oilskin trousers, open neck bathed immediately
cotton shirts with sleeves rolled up,
plus respirators and rubber boots
Series 3: ordinary khaki drill and open
necked shirts but oilskin under-
pants to protect the genital region
Series 4: ordinary tropical battle dress
CDRE India CC-2 impregnated protective drawers, 2 hours
report 285 43 unimpregnated tropical battle dress,
socks, ankle boots, anklets and light
respirators
5.2.2.5.1 Systemic Poisoning after Vapour Exposure of the Skin. After the
vapour exposures reported by Heinen et al.41 described above, no systemic
toxic effects were observed.
The original reports on the volunteer trials carried out in Australia between
1942 and 1945 are not currently available; however, limited descriptions of
these trials were published by Sinclair in the late 1940s.48–51 The published
material describes the systemic toxicity induced by exposure to SM.48 Meth-
ods of exposure and dosages used were not specified, but 438 men were
exposed and, of these, 320 were exposed to vapour and 118 to liquid SM. No
systemic symptoms were noted in any man who developed a lesion less severe
than erythema from vapour and with a less than 20 cm2 raw area induced by
liquid contamination. One hundred and sixty seven men had injuries of this
nature, and 102 of the remaining 271 reported one or more symptom. The
incidence of systemic signs and symptoms was greatest on the first day after
exposure and fell thereafter, but some symptoms persisted for as long as 45
days. The symptoms recorded were nausea, headache, lassitude, insomnia,
vomiting, anorexia, abdominal pain, diarrhoea, tremor, vertigo, tachypnoea
and an “anxiety state”. Of these, nausea, insomnia and headache were the
most common, but the insomnia could have been caused by irritation and
pain from the skin injuries and may not have been a sign of systemic toxicity
in all cases. Only a few cases of abdominal pain occurred on the first day
after poisoning, most cases presented 1–4 weeks after exposure, and when
presenting later occurred within 0.5–1 hour of eating as diffuse epigastric
pain relieved by alkali. The authors comment that diarrhoea did not appear
to be any different to that in non-exposed personnel, although it was not for-
mally recorded in controls. Vertigo was only recorded in subjects who also
complained of nausea. The “anxiety state” was reported in only a few of the
seriously injured men and resolved quickly once the injury started to heal.
The occurrence of 65% of the systemic effects within 24 hours of exposure
when most of the skin injuries had not progressed beyond erythema may
indicate that the systemic effects are not secondary to the skin injury.
The nature of the skin injury in these volunteers is not described in detail
by Sinclair, but he does describe pricking the surface of blisters to release
fluid during treatment and the illustrations in Goodwin’s descriptions of
these trials show blistering of the skin. This is in contrast to the studies
reported by Heinen et al.41 who reported similar injuries but without blisters.
This may be due to use of different challenge dosages (those used in the Aus-
tralian studies are not currently known) or differences in humidity in the
tropics and in Washington, USA, where Heinen et al. performed the expo-
sures, changing the rate of water loss from and through the skin.
5.2.2.6 Liquid
Early observations clearly indicated that exposure of the skin to the smallest
quantities of liquid SM could produce very severe injury. It was also clear that
the human population contained sub-populations of varying sensitivity to
168 Chapter 5
SM and that exposure to SM could increase the sensitivity of those exposed
to its effects.
Marshall et al.52 reported some exposures to solutions of SM in paraffin
(1, 0.1 and 0.01% w/v). These were larger scale tests than those carried out with
vapour by the same researchers (n = 1629 whites and 84 coloured) and showed
that overall, coloured individuals were less sensitive than whites. No coloured
subject reacted to the 0.1% solution whereas 7.5% of the whites tested did, and
only 15% of the coloureds reacted to the 1% solution, while 68% of the whites
did. This supports the view that coloured skin is less sensitive, but does not
permit the difference to be quantified.
Concerns over the exposure of volunteers who had a high sensitivity to SM
during trials prompted work on the definition of the potency of SM in pro-
ducing injury to human skin. A series of trials was undertaken to define the
potency of SM in people who had never been exposed to SM and those who
had. These trials were reported in a series of Porton reports and CDRE (India)
reports in the 1930s. These reports are not written to modern research report
standards and it is easy to discount the results because not all of the experi-
mental details are clear. However, if read together and in the context of other
reports of the time and slightly later, it is clear that they report a unique set
of studies in large groups of humans, and define the potency of liquid SM in
producing damage to the skin.
The aim of these trials was to define the sensitivity of the “normal” and
previously exposed populations to SM and determine the effects of tempera-
ture and ethnic origin. If the reports are read as a whole it is possible, by
assuming that the same methods are used throughout, to come to some con-
clusions about the potency of SM in causing effects on human skin. As might
be expected from the time when these studies were performed, the purity of
the SM used in these studies is not specified, but was probably the same as
the SM being used in weapons at the time.
Some confusion is also caused in the reports by the use of the term “sen-
sitivity” to mean potency and the use of the same word to describe what is
understood today to be sensitisation caused by previous exposure to SM.
The method used was described in an initial study reported by Fairley.53
A 10 µl drop of a 1 in 10 000 dilution of SM in dry benzene (1 µg; in later
studies different dilutions were used) was applied to the volar aspect of the
forearm using a specifically manufactured pipette. This drop was reported
to spread to an area approximately the same size as a “sixpence”, about 2
cm2. The pipette was not described in the UK reports but in one of the later
Indian reports it is described as being drawn from a glass tube and “stan-
dardised by weighing out a quantity of mercury equal in volume to 0.01 cc”.
This was run down the finely drawn glass tube and the volume marked with
a glass pencil. The pipette was described as more labour intensive to use
than an ordinary pipette but more accurate. Although it was clearly not
possible at the time to control for the effect of the benzene solvent on the
permeability properties of the skin or give an accurate measurement of the
purity of the SM, the results of these experiments provide some of the most
robust estimates of the potency of SM to produce effects in human skin.
The benzene solvent would be expected to simultaneously spread and evap-
orate leaving a thin layer of the less volatile SM on the surface of the skin.
As it does this, the benzene would also solvate and perturb the lipids on the
skin and in its superficial layers, which could change the penetration rate,
and hence the potency of the SM. However, to interpret the results of these
tests it is necessary to assume that these phenomena have no effect on the
potency of SM.
5.2.2.6.1 Potency of SM on Human Skin and Sensitisation by Previous

Exposure. In an early study, 302 workers at the UK defence establishment
at Porton Down had 1 µg SM applied to their forearms in 10 µl of benzene
as described above.53 Of these workers, 25 showed a reaction, 24 of whom
had been previously burned with SM and the other one was described as of
unusually “blonde” complexion. In contrast, there were five individuals with
established histories of SM burns who showed no reaction. The skin injury
of those who did react varied from a severe oedematous erythema followed
by desquamation (five subjects) to a just discernible red patch (six subjects).
These reactions were reported using a scoring system used in subsequent
studies in the UK and India:

E− very faint reaction ( just visible red patch)
E faint reaction (erythema less marked than E+)
E+ mild reaction (erythema without oedema)
E++ marked reaction (oedematous, bright red erythema with subsequent
desquamation)

The authors observed that this gave “sensitivities” of 24/53 in those previ-
ously exposed to SM compared to 1/249 in those who had not been exposed
before. Those who reacted in these tests were retested with solutions of SM
in benzene at different dilutions to determine how sensitive they were.54 The
authors concluded that the sensitive individuals at Porton Station were up to
1000 times more sensitive than “normal” subjects. The numbers used in this
study were small—four and six compared to a control group of nine—but
they stimulated further work to define the potency of SM.
A study of the sensitivity of workers at the Sutton Oak manufacturing
plant55 used a wider range of challenge doses and defined the dose response
range for the E−, E, E+ and E++ responses described previously.53,54 What is
clear from the dose response curves that can be produced from these data
(Figure 5.3), and were reproduced in all subsequent work, was that only the
E− or trace effects produced a complete dose response curve defining 0 and
100% responses. The more severe reactions erythema/vesication (E+ and
E++) were not produced in all subjects even at the highest dose used (20 µg).
In a separate study, data were pooled from tests on volunteers, a set of
tests carried out on SS Somersetshire, new arrivals at the Porton Station
and subjects from the Chemical Warfare School; amounting to a total of 681
170 Chapter 5
Figure 5.3 Comparison

of dose response curves for reactions (indicated on the
graph and described in the text) to SM applied to the skin in 10 µl of ben-
zene in a group of workers from the Sutton Oak manufacturing plant.55
All curves fitted using the equation PR = 1/(1 + 10((log ED50 − log dose) × h)),
where PR is the proportional response and h the Hill slope; using
GraphPad PRISM 6.02. Points represent E− or greater (circles), E
or greater (squares), E+ or greater (triangles) or E++ (diamonds) as
defined in the text. The dataset includes both naive and previously
exposed subjects.
subjects.56 The doses required to produce an effect greater or equal to a

“trace” effect were reported and although the variation in these data is much
larger if all the subjects are included than when only the volunteers are ana-
lysed, the ED50 (effective dose, 50%) estimates are very similar in both anal-
yses, being 2.8 µg cm−2 (2.43–3.25; 95CL) for the volunteers alone and 2.6 µg
cm−2 (1.82–4.9; 95CL) for all of the studies pooled.
Later studies performed at Rawalapindi in northern India aimed to dis-
cover the difference between Caucasian and Indian troops in hot57 and cold58
weather (Figure 5.4). After some preliminary trials,59,60 mass sensitivity trials
were conducted and showed Indian troops to be less sensitive than British
troops in hot weather, although the difference was not as great in cold weather.
In these trials, the same methods were used as in those conducted at Por-
ton Station but the reactions to SM were recorded slightly differently as:

Trace A definite visible response
E erythema
V vesication

In Indian subjects an additional reaction “pigmentation” (labelled “P”)
was recorded. This was described as the skin showing a change in pigmenta-
tion that was more than a trace response but without erythema (Figure 5.5).
Figure 5.4 Reactions

of British troops stationed in India (290 exposed) and Indian
troops (300 exposed) to SM.57 Points represent threshold or greater (cir-
cles), pigmentation or greater (triangles), erythema or greater (squares)
and vesication (diamonds).
No relationship between length of service in India and sensitivity in Cau-

casians could be found in the results from the hot weather study.61
One of the original aims of this work, to define the sensitivity of a “nor-
mal” population that had not been previously exposed to SM, was achieved
by using a large number of subjects. Pooling the data generated by the trials
carried out in the UK and in India enables the definition of a dose response
curve for a trace or faint erythematous response, or a more severe response,
in a large population (Figure 5.6).
The conclusion of the studies carried out in India, that Indian troops were
less sensitive than Caucasians, is similar to conclusions from early US stud-
ies that African Americans were less sensitive than Caucasians.38
172 Chapter 5
Figure 5.5 Results

of mass sensitivity trials in hot (squares)57 and cold (triangles)58
weather for British troops stationed in India (open symbols broken
lines) and Indian troops (closed symbols solid lines). Points are propor-
tional responses of a trace reaction or greater. The results clearly show
skin to be more sensitive in hot than cold weather and British troops to
be more sensitive than Indian troops. Numbers in the trials for British
and Indian troops respectively are 290 and 300 for hot weather condi-
tions and 282 and 299 for cold conditions.
5.2.2.6.2 Sensitisation. The observation from early studies that indi-

viduals who had been previously exposed to SM were more sensitive than
naive individuals is supported by comparisons using the dose response of
naive individuals in the mass sensitivity trials described above and the dose
response curve for subjects previously burned by SM (Figure 5.6). The ED50 is
significantly lower in previously burned subjects than in naive subjects (p <
0.001, F-test) and the probit slope is significantly shallower (p < 0.001, analy-
sis of variance on regression analysis).
The question of whether repeated exposure, as would be expected in an
occupational environment, produced a higher degree of sensitisation was
addressed in a subsequent report describing sensitisation of nine workers
over 3 years.63 None of these workers were burned to “casualty severity”
but increased their sensitivity to SM over the 3 years by several thousand
fold.
This is supported by an experimental comparison made by Moore and
Rockman64 who measured the sensitivity of groups of volunteers who had
been previously burned by SM. Three groups were exposed to solutions of
SM in petroleum ether (4.5 µl) in a similar way to the British and Indian stud-
ies. One group had not been previously burned by SM, one group had been
burned once and the third group burned twice. The results clearly showed a
statistically significant 10-fold increase in sensitivity in those volunteers who
had been burned twice, but no significant change in those who had been
burned only once. However, no evidence of cross sensitisation with nitrogen
mustard was found.
Figure 5.6 Dose

response curves from data pooled from eight studies53,55–60,62 for
not previously exposed (solid circles) and three studies53,55,63 for pre-
viously exposed (open squares) subjects. Doses are micrograms of SM
applied to the skin of the forearm in 10 µl of benzene as described in
the text. Fractions represent the number of responders over the num-
ber tested for each point. The ED50s and fitted curves were calculated
using the equation y = 1/(1 + 10((log ED50 − x) × h)), where h is the Hill slope;
using GraphPad PRISM 6.02. The probit slopes were calculated using
the method of Finney.65 Numbers in parenthesis are 95% confidence
limits and R2 is the correlation coefficient for the fitted curves.
These results together with all those relating to skin exposure to liquid SM
applied in 10 µl of benzene are summarised in Table 5.4.
5.3 Conclusions
SM has been called a vesicant because it produces large pendulous blisters on
human skin, but it also produces similar tissue damage, without the formation
of blisters, to the eyes, gut and respiratory tract. No animal species responds to
SM by producing blisters in this way and this combined with the unique biol-
ogy of human skin reduces confidence in extrapolations from animals to man.
The body of human trials reviewed here is a source of data rarely found in the
toxicology of chemicals in that it provides direct measures of the toxic effects
in humans from exposures where the doses and dosages of SM that produce
effects in human eyes and skin are known with some accuracy. The response
to SM in human skin and eyes would be expected to be highly variable in the
general human population with subpopulations having different sensitivities,
although not all of these have been defined in the available reports. It is clear,
for instance, that ethnic skin types (Caucasian, African American, Asian) have
different sensitivities to SM and that SM is a human sensitiser. The environ-
mental conditions also affect sensitivity of the skin to SM, with increased tem-
perature and humidity reducing the dose necessary to produce effects.
174
Table 5.4 Summary
of ED50 (dose producing effect in 50% of those exposed) and probit slope for any definite effect produced by the
application of SM to the skin of the volar forearm in 10 µl dry benzene.
Location Subjects and conditions Previously exposed Number exposed ED50 (µg) Probit slope Ref.
Sutton Oak, northern Workers in manufacturing No 38 10 (9.6–10.4) 1.6 (−0.6 to 3.7) 55
England No
Porton Down, southern Volunteers No 22–163 5.7 (3.7–8.7) 3.0 56
England All groups No 22–681 5.4 (3.8–7.7) 3.2
Rawalapindi, northern Hot weather conditions No 300 0.9 (0.8–1.0) 4.1 (1.6–6.5) 34
India (Indian troops)
Hot weather conditions No 290 0.71 (0.70–0.72) 5.3 (−0.6 to 11.2)
(British troops)
Cold weather conditions No 299 3.5 (2.6–4.9) 2.5 (1.7–3.2) 58
(Indian troops)
Cold weather conditions No 282 0.98 (0.96–1.00) 4.2 (2.6–5.7)
(British troops)
Sutton Oak, northern Workers in manufacturing Yes 18 1.4 (1.0–1.9) 1.2 (0.7–1.7) 55
England
Not specified Workers in manufacturing Yes 9 0.47 (0.2–1.2) 0.6 (−14.6 to 15.7) 54
(Porton report)
Otawa, Canadaa Volunteers No 45–60 3.1 (1.9–2.2) 4.7 (2.5–7.0) 64
Otawa, Canadaa Volunteers Yes (single burn) 17 1.7 (1.2–2.4) 1.9 (1.4–2.4)
Otawa, Canadaa Volunteers Yes (two burns) 14–33 0.26 (0.25–0.28) 2.1 (1.9–2.3)
a
These doses were given in 4.5 µl of petroleum ether. Numbers are means (95% confidence limits) of the dose estimated using the equation
PR = 1/(1 + 10((log ED50 − log dose) × h)), where PR is the proportional response and h the Hill slope; using GraphPad PRISM 6.02. The probit slope was calculated
using the method of Finney,65 also using GraphPad PRISM 6.02. Where no confidence limit is shown the line was calculated from only two challenge doses.
Chapter 5
Although the studies in man do not cover every exposure scenario, combi-
nation with in silico predictions and existing animal studies will allow good
estimates of the effect of SM in most cases.
It is very unlikely that any further controlled exposures of humans to SM
will take place because modern attitudes towards human volunteer studies
would preclude them, now that SM is known to be a genotoxic carcinogen.
This makes the original research reports of the studies that have been con-
ducted an invaluable source of information that must be preserved to guide
future work in animals and in vitro. It is to be hoped that more of the reports
of human trials with SM and other chemical warfare agents will be made
available for public review in the future.
References
1. F. R. Sidell, A History of Human Studies with Nerve Agents by the UK
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2. B. Papirmeister, A. J. Feister, S. I. Robinson and R. D. Ford, The Sulfur
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4. G. Plunkett, The Armourers Remember: The Experimental Stations
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5. J. L. Willems, Clinical Management of Mustard Gas Casualties, Ann. Med.
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Toxicology, 2005, 214, 198–209.
7. S. J. Fairhall, B. J. A. Jugg, R. W. Read, S. J. Stubbs, S. J. Rutter, A. J. Smith,
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8. J. Jenner and S. J. Graham, Treatment of sulphur mustard skin injury,
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9. P. Brookes and P. D. Lawley, The reaction of mustard gas with nucleic
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878, 1426–1432.
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11. A. S. Warthin and C. V. Weller, The Medical Aspects of Mustard Gas Poison-
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Chemical Warfare, ed. E. B. Vedder, Williams & Wilkins Co., Baltimore
USA, 1925, pp. 125–166.
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14. G. W. Beebe, Lung cancer in World War I veterans: possible relation to
1960, 25, 1231–1252.
15. T. Bullman and H. Kang, A fifty year mortality follow-up study of veterans
exposed to low level chemical warfare agent, mustard gas, Ann. Epide-
miol., 2000, 10, 333–338.
16. R. A. M. Case and A. J. Lea, Mustard gas poisoning, chronic bronchitis,
and lung cancer. An investigation into the possibility that poisoning by
mustard gas in the 1914-18 war might be a factor in the production of
neoplasia, Br. J. Med., 1955, 9, 62–72.
17. S. Wada, Y. Nishimoto, M. Miyanishi, S. Katsuta and M. Nishiki, Malig-
nant respiratory tract neoplasms related to poison gas exposure, Hiro-
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18. A. Yamada, On the late injuries following occupational inhalation of
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19. S. Namazi, H. Niknahad and H. Razmkhah, Long-term complications of
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col., 2009, 5, 191–195.
20. A. Firooz, B. Sadr, S. M. Davoudi, M. Nassiri-Kashani, Y. Panahi and Y.
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Cutaneous Ocul. Toxicol., 2011, 30, 64–68.
21. I. Mann, A study of eighty four cases of delayed mustard gas keratitis
fitted with contact lenses, Br. J. Ophthalmol., 1944, 441–447.
22. M. Balali-Mood, H. Kahrom, R. Afshari, D. Attaran, R. Zojaji and M. Kam-
rani, Delayed toxic effects of sulfur mustard on upper and lower respira-
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24. Teulieres, Journal de Medecine de Bordeaux, 1917, lxxxviii.
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26. Teulieres and Valois, Archives D’Ophtalmologie, 1916, xxxv, 403.
27. W. E. Hughes, The importance of mustard burns of the eye as judged by
World War 1 statistics and recent accidents, Fasiculus on chemical Medi-
cine, 1945, vol. 1.
28. G. J. Uhde, Mustard gas (dichloroethyl sulphide) burns of human eyes in
World War II, J Ophthalmol., 1946, 29, 929.
29. H. L. Gilchrist and P. B. Matz, The residual effects of warfare gases, US Gov-
ernment Printing Office, Washington DC, 1933.
30. W. E. Hughes, Mustard gas injuries to the eyes, Ophthalmic Rev., 1942, 27,
582–601.
31. T. J. Phillips, The delayed action of mustard gas and the treatment, Proc.
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32. C. I. Reed, The minimum concentration of dichlor-ethylsulphide (Mus-
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77–80.
33. W. J. F. Guild and K. P. Harrison, The effects of mustard gas on the eyes,
Porton Report 2297, 1941.
34. J. S. Andersen, The effect of mustard gas vapour on eyes under Indian hot
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35. K. Ghabili, P. S. Agutter, M. Ghanei, K. Ansarin and M. M. Shoja, Mustard
gas toxicity: the acute and chronic pathological effects, J. Appl. Toxicol.,
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Mil., 1989, 3, 1–61.
37. J. P. Petrali, S. B. Oglesby, T. A. Hamilton and K. R. Mills, Compara-
tive Morphology of Sulfur Mustard Effects in the Hairless Guinea-Pig
and A Human Skin Equivalent, J. Submicrosc. Cytol. Pathol., 1993, 25,
113–118.
38. E. K. Marshall, V. Lynch and H. W. Smith, On dichlorethylsulphide (mus-
tard gas) II. Varaitions in susceptibility of the skin to dichlorethylsul-
phide, J. Pharmacol. Exp. Ther., 1918, 12, 291–301.
39. S. M. Nagy, C. Golumbic, W. H. Stein, J. S. Fruton and M. Bergmann, The
penetration of vesicant vapors into human skin, J. Gen. Physiol., 1946, 29,
441–469.
40. The effect of mustard vapour on the skin under hot weather conditions, CDRE
(India) Report 245, 1942, UK National Archive Number WO 189/3236.
41. J. H. Heinen, H. W. Carhart, W. H. Taylor, B. N. Stalp, J. C. Connor and N.
M. Clausen, Chamber tests with human subjects. IX Basic tests with vapor,
NRLR P-2579, 1945.
42. W. H. Taylor, H. W. Carhart and L. E. Daily, Chamber tests with human
subjects: I Design and operation of chamber. II Initial tests of navy issue pro-
tective clothing against H vapor, NRLR P-2208, 1943.
43. Report on two cases of severe skin burns from mustard gas vapour under
tropical conditions in India, CDRE (India) Report 285, 1944, UK National
44. B. Renshaw, Observations on the role of water in the susceptibility of
human skin to injury by vesicant vapors, J. Invest. Dermatol., 1947, 75–85.
45. Chemical sampling of CW agents in field experiments, Porton Memo-
randum 19, 1943, UK National Archive Number WO 189/236.
46. W. H. Taylor, H. W. Carhart, L. E. Daily, W. C. Lanning, P. Borgstrom and
A. H. Van Keuren, Chamber tests with human subjects. I. Design and Oper-
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vapor, NRLR P-2208, 1943.
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UK National Archive Number WO 189/2300.
178 Chapter 5
48. D. C. Sinclair, The clinical features of mustard-gas poisoning in man, Br.
Med. J., 1948, 2, 290–294.
49. D. C. Sinclair, The clinical reaction of the skin to mustard gas vapour, Br.
J. Dermatol. Syph., 1949, 61, 113–125.
50. D. C. Sinclair, Treatment of skin lesions caused by mustard gas, Br. Med.
J., 1949, 1, 476–478.
51. D. C. Sinclair, Disability produced by exposure of skin to mustard-gas
vapour, Br. Med. J., 1950, 1, 346–349.
52. E. K. Marshall, V. Lynch and H. W. Smith, On dichlorethylsulphide II vari-
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Exp. Ther., 1919, 12, 291.
53. A. Fairley, Sensitivity to mustard gas, Porton Report 930, 1931, UK National
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55. A. Fairley, Report on the test of ″Sutton Oak″ personnel to determine their
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60. P. S. Richie and S. Anderson, Report on preliminary experiments carried
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65. D. J. Finney, Probit Analysis, 2nd edn, Cambridge University Press,
Cambridge, 1952.
Chapter 6
Long-Term Effects of the

Chemical Warfare Agent
Sulfur Mustard
Kai Kehe*a, Dirk Steinritzb, Frank Balszuweitb,
and Horst Thiermannb
a
Bundeswehr Medical Service Academy, Military Medical Science and
Capability Directorate, Neuherbergstraße 11, 80937 Munich, Germany;
b
Bundeswehr Institute of Pharmacology and Toxicology,
Neuherbergstraße 11, 80937 Munich, Germany
*E-mail: kaikehe@bundeswehr.org
6.1 Introduction
Chemical warfare agents (CWAs) have been used in several conflicts. During
World War I (WWI) a significant quantity of arsenicals, blister and pulmonary
agents was released, causing a tremendous number of casualties. CWAs were
allegedly used in many conflicts after WWI. The last military use between
nations was the Iran–Iraq war (1980–1988), in which sulfur mustard (SM)
in particular was released on a large scale. Approximately 100 000 victims of
CWA use received medical treatment during this conflict.
The military use of CWAs is banned and nearly all nations have signed the
Chemical Weapons Convention. Therefore, the use of CWAs in a war becomes
very unlikely. However, the situation has changed with respect to asymmetric

179
180 Chapter 6
war scenarios or civil wars. Sarin was released in Syria in 2013. The target was
an unprotected civil population with limited access to medical care. Sarin is
a deadly poison and killed most victims. If SM had been used, the situation
would have been different. The limited medical care would have resulted in
a tremendous number of victims with delayed wound healing and develop-
ment of late effects that would have remained even decades after the event.
6.2 Sulfur Mustard

SM is an alkylating agent whose acute effects have been described in detail.1–4
The insight into the pathophysiology of SM poisoning has deepened and
partially has been linked to events at the molecular level.2–4 However,
research on the toxicology of SM poisoning has more or less focused on the
acute effects during the last century. Great efforts have been made to iden-
tify the toxicodynamic processes and subsequent medical countermeasures
against acute poisoning. Unfortunately, no antidote has been identified so
far. The main success has been to limit the tissue damage induced. Taking
into account that SM poisoning has a low mortality, we still face a huge com-
munity of patients with long-term effects. Nowadays most of the patients are
Iranian veterans from the Iran–Iraq war (1980–1988). Approximately 30 000
veterans are still under medical supervision and are a substantial burden on
the Iranian medical system.
SM alkylates a broad range of biomolecules in the human body. Thus,
SM exerts its toxicity both through its primary effect, i.e. DNA and protein
alkylation, and secondary mechanisms, including excessive inflammation,
formation of radical oxygen and nitrogen species, and PARP over-activation
(Chapter 4 in Volume 2). Alkylation of DNA is considered to be the most sig-
nificant cell injury.5 It has been shown in vitro that alkylation of guanine bases
at N7 is the most frequent adduct resulting in the formation of N7-[2-[(2-hy-
droxyethyl)thio]ethyl]guanine (N7-HETEG), followed by the inter- and intra-
strand di-adduct between two guanine bases, resulting in bis[2-(guanine-7-yl)
ethyl]sulfide (Bis-G), N3-[2-[(2-hydroxyethyl)thio]ethyl]adenine (N3-HETEA)
was shown to be another major adduct. These adducts can be found in all
tissues even weeks after cutaneous exposure of mice to SM6 and can possi-
bly account for the development of late effects, including cancer, in these
affected tissues. In addition, fishermen who had been exposed to SM-filled
bomb shells showed a great increase in sister chromatin exchange rates.7–9
Recently, epigenetic changes after SM poisoning were proposed to account
for late effects.10,11 Perturbation of the epigenetic code may explain the long-
term effects, which cannot be explained by SM-induced mutations alone.
SM causes injury via four major routes: (i) skin damage after absorption
through the integument; (ii) eye damage after exposure to mustard gas vapor;
(iii) broncho-pulmonary effects after inhalation; and (iv) systemic toxicity
after ingestion or absorption of high amounts of SM.12
In line with these potential routes, a clinical study revealed that the
delayed toxicity of SM affected the lungs (78%), brain (45%), skin (41%)
Long-Term Effects of the Chemical Warfare Agent Sulfur Mustard 181
and eyes (36%) of 236 Iranian veterans between 2 and 28 months after
exposure.13 Additionally, Khateri et al. investigated a population of 34 000
Iranian veterans 13–20 years after SM exposure. The late effects were diag-
nosed in the lungs (42.5%), eyes (39.3%) and skin (24.5%).14 It is important
to note that pulmonary late effects are the greatest challenge to the medical
system. Table 6.1 gives a summary of the long-term effects caused by SM
exposure.
6.2.1 Cutaneous Injury

In the majority of cases, skin contact with SM induces blistering and necro-
sis with separation of the epidermal and dermal layers. The tissue shows
signs of severe inflammation.15 Later, the skin becomes dry and sensitive
with persistent pruritus, burning and desquamation.13,16 Lesions charac-
terized by erythema and edema without vesication and ulceration usually
do not result in delayed dermal effects.1 Additionally, abnormal pigmen-
tation (hyper- and hypo-pigmentation) can be seen in formerly exposed
areas (Figure 6.1). This form of chemically acquired poikiloderma is
observed in skin areas formerly exposed and additionally sensitive to SM.
These are, for example, in the axillae but not in the palmar region. The sur-
vival of melanocytes as well as their continuous activation account for the
landscape-like pigmentation of the affected skin areas: melanocyte death
Table 6.1 Overview of sulfur mustard-induced long-term effects.

Affected area Long-term effects
Skin 1. Poikiloderma, pigmentation disorders
2. Scarring
3. Eczema
4. Dry skin conditions
5. Prurigo
6. Cherry-like hemangioma
7. Hair loss in affected areas
8. Telangiectasis
Eyes 1. Chronic conjunctivitis
2. Corneal opacification
3. Corneal ulcerations and erosions
4. Stromal scarring
5. Limbal pigmentation
6. Limbal stem cell deficiency
7. Neovascularization (torted vessels)
Respiratory tract 1. Bronchiolitis obliterans
2. Bronchial stenosis
3. Neovascularization and tracheal vulnerability
4. Chronic cough with blood sputum
5. Diminished mucociliary clearance
6. Interstitial lung disease
7. Emphysema
182 Chapter 6
Figure 6.1 Poikiloderma

(hypo- and hyper-pigmented areas) on the back of a
patient in the region of the mustard gas exposure. Skin regions pro-
tected by a belt developed fewer late effects.
results in depigmentation, and a chronic inflammatory state results in

melanocyte activation with subsequent hyperpigmentation.17,18 Hyperpig-
mentation (20–55% incidence) seems to occur more often than hypopig-
mentation (5–25% incidence), depending on the degree of melanocyte
injury.19,20 Patients complain about increased susceptibility to sunlight and
the psychological impact of these skin changes (personal observation). Sim-
ilar findings are reported after the application of nitrogen mustard (HN2).21
Furthermore, chronic late effects are characterized by the occurrence of
cherry-like hemangioma and telangiectasia.22 Chronic skin injury is also
characterized by sensitivity to mechanical injury, chronic eczema and sebor-
rheic dermatitis. Hair loss is frequently observed in formerly exposed areas
(Figure 6.2). Histological evaluation of these sites revealed skin atrophy and
loss of hair follicles. Due to individual disposition, excessive scar formation
or even keloid formation may occur in some patients, resulting in difficul-
ties with contractures and deformity (Figure 6.3). Patients often complain
of itching as a result of chronic pruritus. The clinical picture correlates
with altered levels of interleukin (IL)-2 and IL-6.23 Cooling preparations, e.g.
Figure 6.2 Hair

loss as well as hypo- and hyper-pigmented areas in the axillae of a
patient after SM exposure.
“Panahi’s lotion” (1% phenol, 1% menthol), have proven beneficial effects

in mild pruritus, whereas treatment of severe pruritus with pimecrolimus
1% is recommended.24,25 Dryness and pruritus of the skin are chronic symp-
toms that persist and aggravate through the years. In some cases, hypersen-
sitivity against SM was reported.26 Interestingly, a second exposure to SM
induced a flare-up of old lesions in these patients. These observations are
in line with one of the first reports of SM.27 Some patients report paresthe-
sia in formerly affected regions. Darchini-Maragheh et al. reported impaired
electromyography and nerve conduction velocity in 16.3% of SM exposed
patients even three decades after the initial exposure.28
6.2.2 Ocular Injury

The eyes are the organs most sensitive to SM injury. Acute effects are con-
junctivitis, with dry eye, pain and photophobia. More than 60% of eye inju-
ries recover and vision is restored. The remaining patients develop chronic or
delayed-onset mustard gas keratopathy (MGK). Khateri et al. (2003) reported
that 35% of injured veterans had mild ocular injury (dry eye, conjunctival
scarring and decreased visual acuity) and 3.6% had moderate injury that
included some corneal involvement. Severe symptoms were described in
fewer than 1% of patients.14 MGK is characterized by chronic blepharitis,
meibomian gland dysfunction, dry eye, perilimbal conjunctival ischemia,
184 Chapter 6
Figure 6.3 Extensive

scar formation on the arm of a patient after mustard gas
exposure. The skin is also hypopigmented and there is less hair in the
affected areas.
stem cell deficiency, epithelial irregularity, recurrent or persistent epithe-

lial defects, corneal neovascularization, tortuous blood vessels, stromal
scarring, and secondary degenerative changes including lipoid and amyloid
deposition (Figure 6.4).29,30 MGK develops between 15 and 20 years after
exposure.31,32 The pathology of these changes is not clear. Autoimmune-
related mechanisms have been suggested, with SM adducts and crosslinks in
corneal proteins.33 Stem cell damage as well as epigenetic changes seem to
be more important. The loss of limbal stem cells leads to poor regeneration
of the cornea and even the limbal blood supply is diminished. The result-
ing limbal stem cell deficiency (LSD) accounts for most chronic late effects.13
Therapies for SM-induced ocular injury include tarsorrhaphy, amniotic mem-
brane transplantation, and corneal as well as limbal stem cell transplanta-
tion.30,34,35 In some patients, delayed ulcerative keratopathy was described,
which can result in loss of vision.36
Figure 6.4 Late

eye effects after sulfur mustard exposure include chronic con-
junctivitis, perilimbal hyperpigmentation and late-onset ulcerative
conjunctivitis.
6.2.3 Pulmonary Injury

The respiratory system is very sensitive to SM vapor, which can lead to per-
sistent lung damage and pulmonary disabilities that persist from the acute
to the chronic phase without interruption.37 Delayed respiratory injury is the
major cause of morbidity following exposure to SM. Up to 43% of 34 000 Ira-
nian veterans suffer from lung lesions.14 Most of the patients complain of
chronic cough, dyspnea and sputum production.38 Additional symptoms can
include chest pain, gastro-oesophageal reflux pain and haemoptysis.
Late pulmonary effects after SM injury include obstructive and restric-
tive lung disease. Ghanei and Harandi described the SM-induced changes
as “Mustard Lung”,38 which is characterized by the common symptoms of
chronic obstructive lung disease (COLD): sputum production, shortness of
breath and a productive cough. Additionally, a reduced ratio (75%) between
forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC)
confirms the diagnosis. In SM-induced COLD, bronchiectasis, air trapping in
expiration and mosaic parenchymal attenuation are indicative of a diagnosis
of bronchiolitis obliterans in this population, which is now considered to
be the major process in SM-related lung injury. Bronchiolitis obliterans is
defined by an inflammatory obstruction and consecutive chronic scarring
of the bronchioles. Ghanei et al. confirmed this diagnosis by the use of high-
resolution CT scanning, broncho-alveolar lavage and open lung biopsies.39,40
186 Chapter 6
Restrictive lung disease (RLD) has a high prevalence and has—beside SM
injury—a myriad of causes. It is defined as a reduction in FEV1 and FVC, as
well as a reduction in total lung capacity (TLC; <20% of its normal value).
RLD may have specific causes that can be intrinsic or extrinsic. In the case
of SM-induced RLD, chronic persistent bronchitis was reported in 59% of
patients with a single severe exposure. These patients are heavily disabled.37,41
Some studies also reported chronic bronchitis as a common late effect
of pulmonary SM exposure. Pulmonary fibrosis was found in SM-exposed
Iranian soldiers and can result in a severe impairment of lung function.19
Analysis of broncho-alveolar lavage fluid from those patients revealed that
inflammatory processes are still active even decades after SM exposure.39,42
Fibrotic remodeling of the lung may also cause severe scars and narrowing of
the bronchial system, which can also cause severe health effects.
Ghanei and Harandi recommend the use of corticosteroids in all forms of
SM-induced chronic lung injury. However, side effects of their continued use
may limit their long-term use. Additionally, beta-agonists and anticholiner-
gics are effective in improving lung function.38
6.2.4 Cancers
SM is an alkylating agent that attacks nearly all cell constituents. Alkylation
of DNA is believed to be the most significant event in acute and chronic injury.
It has been shown that alkylation of guanine bases at N7 is the most frequent
adduct, resulting in the formation of N7-HETEG, followed by the inter- and
intra-strand di-adduct between two guanine bases, resulting in Bis-G, and
N3-HETEA has been shown to be another major adduct.43–46 It is believed
that this DNA damage leads to increased rates of cancer in those exposed.
However, although an increase in malignancies appears logical and is
thus assumed, supporting this assumption with reliable scientific and
epidemiological data is challenging. Zafarghandi et al. investigated nearly
8000 veterans who were selected randomly from a cohort of surviving Ira-
nian male soldiers who had at least one proven SM exposure.47 The control
group consisted of nearly 8000 non-exposed comrades. Zafarghandi et al.
detected 84 cancer cases in the exposed population and 49 cases in the con-
trol group. The authors calculated a hazard ratio of 2.02 for the risk of SM-
induced cancer. Although age-specific incidence rates for cancer were found
to be slightly higher in the exposed population than in the unexposed, the
authors found no clear evidence of a higher age-adjusted incidence rate for
specific cancer types that have been previously shown to be associated with
SM exposure. Shortcomings of the study include the missing information
about cohort stratification and the fact that the cohort size of 8000 cases
per group represents a small proportion of the more than 100 000 people
exposed in the first Gulf War. The authors recommend a screening program
for exposed persons. Bullman and Kang had comparable results:48 In a 50
year follow-up study of around 1500 World War II Navy soldiers who volun-
tarily participated in mustard gas chamber tests, the authors were unable to
find any increased risk of cause-specific mortality related to levels of mus-
tard gas exposures that were sufficient to cause skin reactions. However, the
authors did not consider confounding factors (e.g. smoking and drinking
habits), which makes interpretation of the presented results challenging.
Despite these studies indicating no carcinogenic effect, SM is listed as a car-
cinogenic compound by the International Agency for Research on Cancer.
This effect was demonstrated in 245 workers exposed to low levels of SM.
Malignant tumors, especially bronchial carcinoma, bladder carcinoma and
leukemia were reported in this population.49 Takeshima et al. investigated 12
patients with lung cancer after low-level SM exposure. They found that 50%
of lung cancers from SM workers contained somatic point mutations: dou-
ble G:C to A:T transitions, G:C to T:A transversions, A:T to G:C transitions
and single base deletions in the p53 tumor suppressor gene.50 The situation
is different for veterans exposed to a single, high dose of SM. The rate of
lung cancer seems to be slightly increased in WWI veterans.51,52 However,
these studies have some limitations and there is controversy around the car-
cinogenic effect of a single low or high dose exposure, which is typical of
a chemical warfare situation.53 A retrospective study performed by Emadi
et al., who investigated late cutaneous complications after exposure to SM
and nerve agents, revealed a significant increase in malignant skin tumors
(1.9%) in the SM exposure group compared with the control group (none).54
On the basis of the long-term follow-up of former workers in the poisonous
gas factory, Doi et al. concluded that SM decreases the age at which people
are at risk of developing lung cancer.55 Based on our current knowledge, the
development of malignancies cannot be ruled out. Thus, preventive screen-
ing programs for SM-exposed persons should be used.
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Chapter 7
Toxicokinetics of Sulfur
Mustard
Jan P. Langenberga, Marcel J. van der Schansa,
and Daan Noort*a
a
TNO Defence, Safety, and Security, Rijswijk, The Netherlands
*E-mail: daan.noort@tno.nl
7.1 Introduction
Toxicokinetics is the quantitation of the time course of toxic substances in
the body during the processes of absorption, distribution, biotransformation
and excretion. The term originates from the Greek language: toxicon (‘poi-
son’) and kinesis (‘movement’).
Toxicokinetic studies provide a quantitative basis for developing strat-
egies to improve pretreatment and therapy against intoxications with
chemical substances. In the chemical warfare agent domain, toxicokinetic
studies have been performed over the past three decades, and have pro-
duced some unexpected results. For instance, it was long assumed that
due to their high reactivity, nerve agents would disappear nearly instanta-
neously after entering the body. However, Benschop et al. (1987)1 showed
that the nerve agent soman appeared to be surprisingly persistent in the
rat and other animal species. Furthermore, the various stereoisomers of
this nerve agent were found to differ considerably with respect to their
toxicokinetics and toxic potency. This finding has provided guidance for

191
192 Chapter 7
the development of medical countermeasures against soman and other
nerve agents.
Another example is the difficulty of treating percutaneous intoxications
with VX in laboratory animals.2 This was unexpected since acetylcholin-
esterase, which is inhibited by VX, is easily reactivated by oximes in vitro.
Also, intoxications with subcutaneously administered VX could easily be
treated with oximes.3 Van der Schans et al. (2003)4 showed that VX is highly
persistent in hairless guinea pigs after percutaneous administration, since
the agent is slowly absorbed via the skin and not rapidly detoxified. Joosen
et al. (2010)5 reported that the difficulty of treating a percutaneous intox-
ication is due to a mismatch between the pharmacokinetics of the nerve
agent antidotes after single or triple intramuscular administration and the
toxicokinetics of the percutaneously administered VX; in other words, the
nerve agent keeps entering the body after the oxime has been cleared from
the body, thus re-inhibiting the reactivated acetylcholinesterase. The only
way to effectively treat a severe percutaneous VX poisoning is by repeated
administration of the antidotes upon (re)appearance of the signs and
symptoms of intoxication.
With regards to the toxicokinetics of nerve agents, it should be pointed
out that this subject has been addressed extensively in the recent past.
Firstly, Van der Schans et al. (2008)6 reviewed the fate of the individual ste-
reoisomers of intact VX and various G agents. It appeared feasible to ana-
lyze nerve agents in several biological matrices at a toxicologically relevant
level, and to determine the time window during which acutely toxic levels
exist. The most remarkable finding was that the persistence of nerve agents
is much more pronounced than expected on purely chemical grounds. This
is important for the development of novel countermeasure strategies, such
as the application of bioscavengers. Secondly, John et al. (2015)7 recently
reviewed the toxicokinetics of both nerve agents and vesicants, and focused
in particular on the absorption, distribution, metabolism and excretion
(ADME) aspects of these agents, including the bio-analytical techniques
required for measurement of the intact agent, its resulting metabolites
and adducts. One of the key messages was that whereas the metabolism
of nerve agents is rather straightforward and binding seems to be limited
to only a limited number of sites at toxicologically realistic levels, the met-
abolic fate and binding characteristics of sulfur mustard appeared to be
much more complex.
So, observing that the toxicokinetics of intact nerve agents, and ADME
aspects of both nerve agents and sulfur mustard have been covered in enough
detail, we decided not to address these subjects in the current chapter, in
order to avoid redundancy. A thorough review of the fate of intact sulfur
mustard is still lacking, however, and consequently the current chapter will
focus on the toxicokinetics of sulfur mustard in a variety of animal species
and administration routes. In addition, the toxicokinetic aspects (including
ADME) of sulfur mustard as observed during a number of accidental human
exposures will be addressed.
Toxicokinetics of Sulfur Mustard 193
7.2 Experimental
7.2.1 Analytical Procedures
In order to study the toxicokinetics of sulfur mustard, a method to deter-
mine the compound in biological matrices (blood and tissues) is needed,
preferably with a sensitivity that allows quantitative analysis below the level
of toxicological relevance. In several studies,8–11 14C-labeled sulfur mustard
has been used, which allows for convenient and sensitive analysis, but since
only radioactivity is measured, the identity of the measured compound(s) is
not confirmed, which may be considered a drawback of this approach.
Sulfur mustard can be extracted from blood and tissue samples via
liquid–liquid or solid phase extraction (SPE). The absolute recovery of sul-
fur mustard via liquid–liquid extraction with ethyl acetate appeared to be
higher (80–90%) than with SPE (ca. 70%).12 Furthermore, the selectivity
of SPE was no better than that of liquid–liquid extraction. The addition of
sodium chloride, advocated by Maisonneuve et al. (1993)13 to avoid hydroly-
sis to hemi-mustard and thiodiglycol, did not appear to be necessary. Fully
deuterated (d8) sulfur mustard appeared to be an excellent internal standard,
since the recovery of sulfur mustard relative to the deuterated internal stan-
dard was 99% under all extraction conditions. No problems with respect to
the stability of sulfur mustard in blood samples were encountered when the
samples were extracted immediately after being drawn from the animal.
Being a relatively volatile compound, sulfur mustard can be analyzed in
biological materials using gas chromatography (GC) combined with flame
ionization (FID; used by Maisonneuve et al. 1993 13), flame photometric detec-
tion (FPD; used by Zhang and Wu, 1987 14), electron capture detection (ECD),
chemoluminescence detection (CSD) or mass spectrometry (MS). The sensi-
tivity and selectivity of the various detection principles has been compared
by Langenberg et al. (1998).12 FID is simple and robust but not very selective
and relatively insensitive. The absolute detection limit (signal to noise ratio = 3)
of GC-ECD for sulfur mustard was 17 pg, but the stability of the detector
was problematic. SCD was about four times less sensitive than ECD, but
the selectivity was much higher. FPD was highly selective, but one order of
magnitude less sensitive than ECD. ‘Pulsed’ FPD (PFPD) offered both a high
selectivity and a sensitivity comparable to that of ECD. The most sensitive
detection method for sulfur mustard appeared to be MS. In the semi-single
ion mode at (m/z 109 for sulfur mustard) an absolute detection limit of ca.
700 fg was routinely reached. The ability to confirm the identity of the ana-
lyzed compounds was considered to be an important advantage. In the past
two decades, MS detectors have been improved further. In particular, the
availability of GC-MS/MS systems increased the sensitivity of target analysis
significantly. However, this technology was not available in the days that the
toxicokinetic studies of sulfur mustard reported here were performed. If the
toxicokinetic studies were performed in this day and age, GC-MS/MS would
be the preferred analytical technique. In order to be able to analyze very low
194 Chapter 7
blood or tissue levels of sulfur mustard, as much of the sample extract as
possible needs to be injected into the GC. Maisonneuve et al. (1993)13 accom-
plished this by concentrating the equivalent of 6 ml of blood into 100 µl of
ethyl acetate, of which 1 µl was injected on-column into the GC-FID configu-
ration (corresponding to 60 µl of blood). Their actual detection limit was ca.
40 ng ml−1 of blood. Langenberg et al. (1998)12 chose another approach: the
injection of up to 500 µl of the ethyl acetate extract (corresponding to 0.5 ml
of blood) into the GC via thermal desorption from Tenax TA. Due to the high
sensitivity of MS detection the actual detection limit was 5 pg ml−1 of blood
or better.
7.2.2 Animal Models

The toxicokinetics of sulfur mustard have been studied in various animal
models, e.g., the rat, hairless guinea pig, small pig and marmoset monkey.
The rat is a generally accepted model for toxicokinetic studies. Maison-
neuve et al. (1993)13 do not provide a rationale for choosing this rodent as an
animal model for sulfur mustard intoxication. The authors only studied the
intravenous (iv) toxicokinetics.
Langenberg et al. (1998)12 chose the hairless guinea pig (male, ca. 500–990 g)
in order to study the percutaneous toxicokinetics of sulfur mustard. They
also studied the toxicokinetics for the respiratory route and the iv route,
the latter as a reference route of administration, with 100% bioavailabil-
ity. The hairless guinea pig has been advocated as a suitable small animal
model for studies on the (per)cutaneous toxicology of sulfur mustard.15,16
The epidermis of the hairless guinea pig is thicker than in most other labo-
ratory animals and therefore more similar to the human epidermis. Never-
theless, the cell layers in the hairless guinea pig epidermis are thinner than
those in humans. Furthermore, the stratum corneum of the hairless guinea
pig is considerably thinner than that in humans. This has to be taken into
account when trying to extrapolate the results obtained in hairless guinea
pigs to humans. In this respect, the pig is a better model for humans, since
its skin resembles that of humans more closely. However, the pig is far from
a small laboratory animal. Its use in, e.g. respiratory or percutaneous vapor
studies, is accompanied by technical difficulties. Mini pigs are easier to use
in such studies. Their use in sulfur mustard research has been reported
(e.g. Rice et al. 2000 17), but not (yet) in toxicokinetic studies. A major prob-
lem with the hairless guinea pig since the 1990s was its limited availability.
Over the years this problem has increased, and currently, this animal is
hardly available.
Zhang and Wu (1987)14 chose the small pig (male and female, black,
4.2–13.0 kg) because of the aforementioned resemblance of its skin to that
of humans. They chose this model to study the percutaneous toxicokinetics
of occluded liquid sulfur mustard, and studied the iv route in this species
as the reference route, as well as the toxicokinetics after subcutaneous (sc)
exposure.
The respiratory route is also highly relevant for sulfur mustard. Due to
their complex nasal system, rats and guinea pigs may not be the best models
for humans when studying the inhalation toxicokinetics of sulfur mustard
vapor.18 In this respect, the marmoset monkey, a small non-human primate,
is a better choice. The group of Langenberg et al. studied the inhalation toxi-
cokinetics of sulfur mustard in this species (male and female, ca. 300–500 g),
using the iv route as a reference.19–21
7.3 Toxicokinetics of Sulfur Mustard

7.3.1 Intravenous Toxicokinetics of Sulfur Mustard
The first report on the iv toxicokinetics of sulfur mustard was published by
Zhang and Wu (1987),14 using small pigs and GC-FPD for the analysis of sul-
fur mustard in blood samples. Later studies include those of Maisonneuve
et al. (1993),13 in the rat using GC-FID, and Langenberg et al. (1997, 1998),19,22
in the hairless guinea pig and marmoset, using GC-MS. The concentration–
time profiles for all studies are shown together in Figure 7.1, the calculated
toxicokinetic parameters are presented in Table 7.1.
The iv route is used as a reference in toxicokinetic studies, since the sys-
temic availability for this route is 100%. The systemic availability of more
Figure 7.1 Concentration–time

profiles for iv bolus administration of sulfur mus-
tard to the rat (solid line, 10 mg kg−1), HGP (0.3 LD50/2.46 mg kg−1 and 1
LD50/8.2 mg kg−1), pig (10 mg kg−1) and marmoset (8.2 mg kg−1). The tox-
icokinetic parameters used to draw these curves are presented in Table
7.1. HGP: hairless guinea pig.
196 Chapter 7
Table 7.1 Toxicokinetic
parameters for intravenous administration of sulfur mus-
tard to rats, hairless guinea pigs, pigs and marmosets.a
Hairless Hairless
Parameter Unit Ratb guinea pigc guinea pigd Pige Marmosetf
Dose mg kg−1 10 2.46 8.2 10 8.2
Exponents — 2 2 2 3 3
A ng ml−1 1294 826 4665 52 400 3881
α min−1 0.12 1.97 1.41 1.63 0.32
B ng ml−1 46 0.85 0.78 14 000 142
β min−1 0.0032 0.0061 0.02 0.522 0.048
C ng ml−1 — — — 900 2.4
γ min−1 — — — 0.058 0.0026
AUC ng min 25 088 1294 6686 82 000 14 450
ml−1
C0h ng ml−1 1339 828 4666 67 300 4025
g g
k1,2 min−1 0.063 0.209 0.082
g g
kel min−1 0.053 0.64 0.70
g g
k2,1 min−1 0.0072 0.0081 0.0022
t1/2,disi min 5.8 0.81 0.89 0.43 g
t1/2,el j min 216 114 347 12 267

V1k l kg−1 7.47 2.97 1.76 2.47 2.04
g g
Vdss l kg−1 72.8 79.55 66.07
Cl l min kg−1 0.40 1.90 1.23 0.138 0.57
MRT min 182 41.8 53.9 4.1 31.5
a
General equation: Ct = A × e−α × t + B × e−β × t + C × e−γ × t. Formulae for the toxicokinetic parame-
ters: AUC = A/α + B/β + C/γ, Cl = Dose/AUC, k2,1 = (A × β + B × α)/(A + B), kel = α × β/k2,1, k1,2 = α + β
− k2,1 − kel, C0 = A + B + C, t1/2,dis = ln 2/α, t1/2,el = ln 2/β, V1 = Dose/C0, Vdss = V1(l + k1,2/k2,1), MRT =
(A × (1/α)2 + B × (1/β)2 + C × (1/γ)2)/AUC.
b
Data taken from Maisonneuve et al. (1993).13
c
Data taken from Langenberg et al. (1998).12
d
Data taken from Langenberg et al. (1998).12
e
Data taken from Zhang and Wu (1987).14
f
Data taken from Langenberg and Trap (2005).20
g
Parameter could not be calculated.
h
C0: extrapolated concentration in the central compartment at time 0.
i
t1/2,dis: distribution half-life.
j
t1/2,el: terminal half-life.
k
V1: volume of the central compartment.
relevant routes (in this case the respiratory and percutaneous routes) is cal-
culated relative to that of the iv route. The iv toxicokinetics are described for
various animal species below.
7.3.1.1 Rat
Maisonneuve et al. (1993)13 administered a dose of 10 mg kg−1 to rats, corre-
sponding to three lethal doses, 50% (LD50; 14 days). The concentration–time
profile could be described with a two compartment model, with a very rapid
distribution phase and a relatively slow elimination phase. The calculated
toxicokinetic parameters are listed in Table 7.1.
After iv administration to rats of 14C-labeled sulfur mustard at the same
dose, radioactivity was detected in the blood, plasma, kidney, liver, intestine
and stomach, heart, lung, brain, spleen, eyes, testicle, and adrenal gland.8
From 10 min up to 6 h after administration, the liver and kidney showed
more radioactivity than the blood. The organs with the lowest levels of radio-
activity were the brain, spleen, eye and testicle. The maximum radioactivity
in the organs was reached around 2–3 h after iv administration. The radioac-
tivity peaked in fat and skin at 35 min after administration. The authors also
reported that the observed distribution was influenced by the vein in which
the agent was injected. After injection into the femoral vein, the injected leg
was a site of significant radioactivity distribution, whereas the jugular vein
injection did not result in significant accumulation in the lung. The heart,
lung, brain and spleen received greater proportionate shares of radioactivity
at 35 min after jugular vein injection compared with femoral vein administra-
tion. Since only radioactivity was measured and the identity of the radioac-
tive species was not elucidated, it is unknown to what extent the distribution
of intact sulfur mustard and/or its 14C-retaining metabolites were measured.
7.3.1.2 Hairless Guinea Pig

Langenberg et al. (1998)22 administered a dose of 8.2 mg kg−1 to hairless
guinea pigs ( jugular vein), corresponding to 1 LD50 (96 h). In order to estab-
lish whether or not the toxicokinetics are linear with the dose, the toxi-
cokinetics of a dose corresponding to 0.3 LD50 were also studied. For both
doses, the concentration–time profiles were described best with a two com-
partment model. Both profiles are characterized by a very rapid distribution
phase and a relatively slow elimination phase. The calculated toxicokinetic
parameters are listed in Table 7.1. In the hairless guinea pig, distribution
appears to occur faster than in the rat, whereas elimination proceeds at a
comparable rate. Comparison of the area under the curve (AUC) values cal-
culated for administration of doses corresponding to 1 and 0.3 LD50 indicates
a non-linear relationship between the toxicokinetics and the dose. Usually, a
non-linear relationship between toxicokinetics and the dose are associated
with saturable metabolic pathways. It is not clear what process is responsible
for the non-linear toxicokinetics in the hairless guinea pig. Tentative expla-
nations are covalent binding of sulfur mustard to, for example, glutathione
and albumin.
After correction for the differences in administered dose per kilogram of
bodyweight, the AUC in the rat was 3-fold higher than in the hairless guinea
pig. This could be the result of interspecies variation, but also of non-lin-
earity with the dose. Since the AUC and total body clearance (Cl) are related
parameters, the Cl of sulfur mustard in the hairless guinea pig is ca. 3-fold
higher than in the rat. The rate constants of transfer between compartments
1 and 2 (k1,2) and between compartments 2 and 1 (k2,1) are comparable for
the two species, whereas the elimination rate constant (kel) is 15-fold higher
in the hairless guinea pig than in the rat. Marked differences are observed
between the values calculated for the volume of the central compartment (V0)
and the volume of distribution under steady-state (Vdss) for the two species.
198 Chapter 7
Furthermore, there is a 25-fold difference in the mean residence time (MRT).
This parameter represents the mean lifetime expectancy of each sulfur mus-
tard molecule in the body.
At 3 and 10 min after iv administration of a dose corresponding to 1 LD50
the concentrations of intact sulfur mustard in the lung, spleen and bone
marrow were higher than in the blood. In the liver, the concentration of
agent was about equal to that in the blood at 3 min, but had increased about
3-fold at 10 min after administration. At 3 and 6 h the concentration of sulfur
mustard in the lung was approximately equal to that in the blood, whereas
the concentrations in the spleen, bone marrow and liver were one to two
orders of magnitude higher than in the blood. These results indicate a sub-
stantial partitioning of sulfur mustard from the blood to the tissues. At 10
min after administration of a dose corresponding to 0.3 LD50, the concentra-
tions of sulfur mustard in the liver, lung, spleen and bone marrow exceeded
that in the blood. However, at 2 h after administration, only the concentra-
tions of intact agent in the liver and bone marrow exceeded that in the blood,
whereas at 4 h this was only the case for the liver.
The concentration time course of the major adduct of sulfur mustard to
DNA, N7-(2-hydroxyethylthioethyl)-2′-deoxyguanosine in the various tissues
followed the general pattern of that of intact sulfur mustard in these tissues.
By far the highest concentration of N7-(2-hydroxyethylthioethyl)-2′-deoxygua-
nosine was observed in lung tissue. After administration of a dose corre-
sponding to 1 LD50, the adduct level in the lung was already high at 3 min
after administration and remained high for up to 48 h after administration.
Remarkably, the adduct level in the blood was almost 30-fold lower than in
the lung, as early as 3 min after iv administration. At 48 h after administra-
tion, the adduct level in the blood was ca. 1 per 107 nucleotides, which was
still ca. 20-fold lower than in the lung. This observation can at least partly be
explained by the extensive partitioning of sulfur mustard from the blood into
the lung. In addition, a higher accessibility of DNA to sulfur mustard in lung
cells than in white blood cells cannot be excluded. In the spleen, the concen-
tration of N7-(2-hydroxyethylthioethyl)-2′-deoxyguanosine increased in the
period of up to 3 h after administration. After 24 h the number of adducts
had decreased to ca. 1 per 107 nucleotides. In the bone marrow the adduct
level showed some variation in the first 6 h, after which the concentration
of N7-(2-hydroxyethylthioethyl)-2′-deoxyguanosine gradually decreased. The
adduct level in the liver appeared to be maximal at 3 h after administration.
The level had decreased at 6 h, whereas the adducts had nearly disappeared
at 24 h. In the small intestine, a fast accumulation of adducts during the
first 10 min after administration was observed. The adduct level subse-
quently decreased, but remained at a steady level of about 2 adducts per 107
nucleotides for up to 48 h after administration. The concentrations of N7-(2-
hydroxyethylthioethyl)-2′-deoxyguanosine in tissues after iv administration
of a dose corresponding to 0.3 LD50 were more than 3.3-fold lower than those
measured for 1 LD50, underscoring the non-linearity of the toxicokinetics
with the dose. At 10 min after administration of 0.3 LD50 sulfur mustard,
7
the highest adduct levels were found in the blood (5 adducts per 10 nucle-
otides) and the lung (ca. 3 adducts per 107 nucleotides), whereas the levels
were ca. 0.5 adducts per 107 nucleotides in the spleen and small intestine. In
the bone marrow, no adducts were detectable at 10 min after administration
of this dose. At 48 h after administration, the adduct had largely disappeared
from the lung, spleen, bone marrow and small intestine, whereas the level
in the blood was only slightly lower. Interestingly, the concentration of N7-(2-
hydroxyethylthioethyl)-2′-deoxyguanosine in the lung did not reach such
extreme values as was observed after administration of a dose corresponding
to 1 LD50. This finding is in agreement with the relatively low concentration
of intact sulfur mustard in lung tissue after iv administration of a dose corre-
sponding to 0.3 LD50. However, the concentration of N7-(2-hydroxyethylthio-
ethyl)-2′-deoxyguanosine in the blood was the same order of magnitude for
both doses shortly after administration of sulfur mustard. This suggests that
a substantial amount of sulfur mustard will covalently bind immediately in
the blood, which is a saturable process.
7.3.1.3 Pig
Zhang and Wu (1987)14 administered an iv dose of 10 mg kg−1 bodyweight
to the pig. The rationale behind this dose is not revealed in the paper. The
concentration–time profile of sulfur mustard in the blood could be mathe-
matically described as a three compartment model, with a very fast initial
distribution phase and a rapid elimination half-life. The AUC is about 3 times
higher in the pig than in the rat for the same dose. The calculated toxicoki-
netic parameters are listed in Table 7.1.
7.3.1.4 Marmoset
Langenberg et al. (1997)19 administered a dose of 8.2 mg kg−1 to marmosets
(via the jugular vein). This dose corresponds to 1 LD50 (96 h) in the hairless
guinea pig. The LD50 was not determined in the marmoset. The concentra-
tion–time profile was described better with a tri-exponential equation than
with a bi-exponential equation, as determined with the F-ratio test.23 The cal-
culated toxicokinetic parameters are listed in Table 7.1. The terminal half-
life of sulfur mustard in the marmoset is comparable to that in the hairless
guinea pig at the same dose. Due to the slower distribution in the marmoset,
the AUC is more than 2-fold larger than in the hairless guinea pig. Conse-
quently, the Cl in the hairless guinea pig is twice that in the marmoset.
The concentrations of intact sulfur mustard in marmoset tissues were not
measured, but the concentrations of N7-(2-hydroxyethylthioethyl)-2′-deox-
yguanosine were. For all marmoset tissues studied (lung, liver, spleen, bone
marrow and intestines) the N7-(2-hydroxyethylthioethyl)-2′-deoxyguanosine
concentrations were lower than in the hairless guinea pig after adminis-
tration of 8.2 mg kg−1, indicating that less partitioning from the blood to
200 Chapter 7
the tissues occurs in the marmoset. As in the hairless guinea pig, by far the
highest adduct concentration was found in the lung tissue of the marmosets.
Hardly any adduct was measured in the marmoset liver, in contrast to the
hairless guinea pig.
7.3.2 Subcutaneous Toxicokinetics of Sulfur Mustard

7.3.2.1 Pig
Zhang and Wu (1987)14 studied the toxicokinetics of sulfur mustard after sc
administration of a dose of 200 mg kg−1 to the pig. Concentrations in arterial
blood (carotid artery) were measured up to 90 min after administration and
were ca. 360 ng ml−1 at maximum, around 25 min after administration of
the agent. The measured concentration–time course could be fitted to a one
compartment model with first-order absorption and elimination, see Figure
7.2. The toxicokinetic parameters calculated from the curve fitting equation
are shown in Table 7.2.
The systemic bioavailability for the sc administration route (Fsc) was cal-
culated using the iv route as the 100% reference, with the formula:

AUCsc Div
= Fsc × (7.1)
AUCiv Dsc

Figure 7.2 Concentration–time

course of sulfur mustard (± standard deviation) in
arterial blood of pigs (n = 3) after sc administration of 200 mg kg−1. Data
taken from Table 6 of Zhang and Wu (1987).14
parameters for sc administration of sulfur mustard to pigs
(n = 3).a
Parameter Unit Pig
−1
Dose mg kg 200
Exponents — 2
A ng ml−1 554.6b
α min−1 0.012b
β min−1 0.093b
AUC ng min ml−1 40 254
Cmaxd ng ml−1 358
tmaxg min 25.2
t1/2,abse min 7.45b
t1/2,elf min 58
F 0.024c
a
General equation: [sulfur mustard] = A(e−α × t − e−β × t).
b
Data taken from Table 6 of Zhang and Wu (1987).14 Other parameters calculated with standard
pharmacokinetic formulae using these data.
c
The bioavailability (F) has been calculated in relation to the data of Zhang and Wu (1987)14 for
iv administration of sulfur mustard to pigs.
d
Cmax: maximum concentration.
e
t1/2,abs: absorption half-life.
f
g
tmax: time of Cmax.
and appeared to be ca. 2.4%. In view of the relatively high lipophilicity of sul-
fur mustard it is no surprise that the availability of the agent in the blood is
rather low after injection into the subcutis. The elimination half-life is 5-fold
longer for sc administration than for the iv route. It may be that slow absorp-
tion is still occurring at the injection site in the phase that is considered to
reflect elimination.
7.3.3 Inhalation Toxicokinetics of Sulfur Mustard

7.3.3.1 Rat
Benson et al. (2011)11 exposed F344 rats via a transorally placed tracheal
catheter to ca. 250 mg m−3 of 14C-sulfur mustard for 10 min, after which the
radioactivity was measured in various tissues at several time points up to
7 days after ending the exposure. By exposing the animals in this way the
absorption of sulfur mustard in the upper airways was circumvented. At 2
h after ending the exposure, more than 70% of the inhaled body burden
was located in the carcass, pelt and intestines. The remainder of radioac-
tivity was located in the liver, lungs and blood. Radioactivity in these tis-
sues decreased with time, whereas that in the kidney increased up to 7 days
after exposure. Since only radioactivity was measured and the identity of
the radioactive species was not elucidated, it is unknown to what extent the
distribution of intact sulfur mustard and/or its 14C-retaining metabolites
was measured.
202 Chapter 7

During and after 5 min nose only exposure of anesthetized, restrained hair-
less guinea pigs to 160 mg m−3 sulfur mustard in air, sulfur mustard was
not detectable in the blood, i.e. the concentration was <5 pg ml−1.12,22 How-
ever, this exposure (800 mg min m−3) was determined to correspond to one
lethal concentration, 50% (LCt50) for the hairless guinea pig. Analysis of
the respiratory tract of hairless guinea pigs exposed to this dose at 4 h after
ending the 5 min nose only exposure revealed adducts of sulfur mustard
to DNA mainly in the larynx and trachea, and hardly any in the lungs. The
fact that sulfur mustard could not be detected in the blood may therefore
be explained by the almost exclusive distribution of the agent within the
respiratory tract. Evidently, respiratory exposure of hairless guinea pigs to
sulfur mustard vapor will lead predominantly to local damage in the respi-
ratory tract.
Nose only exposure to 2400 mg min m−3 did result in measurable con-
centrations in the blood, with a maximum of 4.3 ng ml−1. This dose, corre-
sponding to 3 LCt50, could be reached by exposing the animals for 8 min to a
concentration of 300 mg m−3, which was about the maximum that could be
generated dynamically in a controlled way. Toxicokinetic evaluation was only
possible by assuming a very rapid absorption process in combination with a
slow absorption process. Calculated parameters are presented in Table 7.3.
DNA adducts were measurable in the blood, lung tissue and spleen, with the
highest concentrations in the lungs.

parameters for nose only exposure to sulfur mustard vapor
of hairless guinea pigs and marmosets.a
Parameter Unit Hairless guinea pig Marmoset
−3
Dose mg min m 2400 800
b
A ng ml−1 −4.92
b
B ng ml−1 6.44
b
C ng ml−1 320
γ min−1 b
0.467
b
D ng ml−1 0.074
b
δ min−1 0.00246
AUC ng min ml−1 320c 740
t1/2,disd min b
1.5
t1/2,ele min b
280
a
The inhalation results were fitted with a discontinuous function: [sulfur mustard] = A + B × t
for the absorption phase (0–5 or 8 min) and [sulfur mustard] = C × e−γ × t + D × e−δ × t for the
distribution and elimination phase (>5 min).
b
Parameter could not be calculated (no meaningful fit of distribution and elimination phase).
c
AUC calculated with trapezoidal method for t = 0–240 min.
d
t1/2,dis: distribution half-life.
e
7.3.3.3 Marmoset
During and after 5 min nose only exposure of anesthetized, restrained mar-
mosets to 160 mg m−3 sulfur mustard vapor in air, the intact agent was mea-
surable in the blood with a maximum concentration of 29 ng ml−1 at the
end of the 5 min exposure period.20,21 This dose corresponds with 1 LCt50
in the hairless guinea pig (96 h). The LCt50 of sulfur mustard was not deter-
mined in the marmoset. The absorption phase (0–5 min) was described
with a mono-exponential equation, and the post-exposure phase (5–60 min)
with a bi-exponential equation. The concentration–time course is shown in
Figure 7.3, the calculated parameters are presented in Table 7.3. The termi-
nal half-life was calculated to be 280 min, which is very close to the value
of 270 min determined for iv administration of 8.2 mg kg−1 (corresponding
to 1 LD50 in the hairless guinea pig) to the marmoset. The observation that
exposure of hairless guinea pigs to the same dose did not lead to a detect-
able concentration of sulfur mustard in the blood suggests that the marmo-
set is a more suitable animal model to study the inhalation toxicokinetics
of sulfur mustard than the hairless guinea pig, due to the lower complexity
of the upper airways of this non-human primate.
In separate experiments the concentrations of sulfur mustard were mea-
sured in various tissues at several time points, up to 24 h after exposure.
Figure 7.3 Mean

concentration–time course with standard error of the mean (n = 6)
of sulfur mustard in the blood of anesthetized, restrained marmosets
during and after 5 min nose only exposure to 160 mg m−3 sulfur mus-
tard vapor in air.
204 Chapter 7
The highest concentrations of intact sulfur mustard were found in the bone
marrow. The concentration was already high 10 min after starting the expo-
sure (ca. 1.2 ng g−1) and increased within the first hour to ca. 4.5 ng g−1.
At t = 10 min the concentration of sulfur mustard in the bone marrow was
about 3-fold lower than that in the blood, but at t = 60 min it was about 75
times higher. In the liver the concentrations were already high 10 min after
starting the exposure (ca. 0.9 ng g−1), and decreased slowly with time. In fat
tissue the concentration built up in the first hour after the exposure. The con-
centrations in the spleen and lung were very low, and much lower than those
in the blood at the corresponding time points. At 24 h after exposure concen-
trations of intact sulfur mustard were no longer detectable (<10 pg g−1).
The highest DNA adduct concentrations were found in the upper airways
from the nasal mucosa down to the bifurcation. The concentrations in the
lung were very low but higher than those in the spleen, bone marrow, small
intestine and blood.
7.3.4 Percutaneous Toxicokinetics of Sulfur Mustard

7.3.4.1 Pig
Riviere et al. (1995)9 studied the toxicokinetics of topically administered
14
C-sulfur mustard in their isolated perfused porcine skin flap (IPPSF) model
to support the correlation between critical steps in the vesication process and
concentrations of the agent in different skin regions. About 90% of the radio-
activity was not recovered due to evaporation of the agent from the skin. No
attempts were made to avoid evaporation, since occlusion was not considered
to be a realistic exposure condition. Absorption mainly occurred within the
first hour after application, with the majority of the absorbed radioactive dose
either in the skin or venous blood stream of the IPPSF. A large fraction of the
measured radioactivity was probably not intact sulfur mustard; however, the
identity of the radioactive species was not established. A multicompartmental
toxicokinetic model was developed to predict penetration into and distribu-
tion within the IPPSF. The authors report that sulfur mustard decreased the
vascular volume of distribution in IPPSF in a dose dependent way, a phenome-
non that should be incorporated into the toxicokinetic model.
Zhang and Wu (1987)14 studied the toxicokinetics of liquid sulfur mustard
applied at a dose of 200 mg kg−1 on 5 cm2 of the skin of three small black pigs,
followed by occlusion with polyethylene foil and a glass disk. Blood samples
were drawn up to 4 h after application, but no sulfur mustard was detected
in these samples. The pigs showed skin damage as well as symptoms of sys-
temic poisoning.

Logan et al. (1999)10 determined the cutaneous uptake of 14C-sulfur mus-
tard from saturated vapor by the hairless guinea pig using vapor caps
adhered to the dorsal skin. The skin was exposed to the saturated vapor at
16–22 °C and 17–66% relative humidity for 7 min. The uptake rate was cal-
culated to be ca. 2 µg cm−2 min−1. This is in reasonable agreement with the
findings of Renshaw (1946)24, who reported values in the range of 1–4 µg cm−2
min−1 for the rate of penetration of saturated sulfur mustard vapor into human
skin at 21 °C. Bergmann et al. (1945)25 reported the penetration rate of sulfur
mustard vapor applied to human forearms to be 1.4 µg cm−2 min−1 at 21–23 °C
and a relative humidity of 44–46%. This penetration rate was constant in the
time window of 3–30 min. In terms of the uptake of sulfur mustard from vapor,
human and hairless guinea pig skin are, therefore, comparable.
Langenberg et al. (1998)22 exposed the whole body of anesthetized,
restrained hairless guinea pigs to 238 mg m−3 for 45 min. The animals were
breathing clean air and therefore were not exposed via inhalation. The expo-
sure of ca. 10 000 mg min m−3 was estimated to correspond to approximately
1 LCt50, based on data from the literature.16 During exposure the concentra-
tion of sulfur mustard in the blood gradually increased to 12 ng ml−1. The
observed fairly high percutaneous absorption rate was anticipated because
of the rapid cutaneous uptake reported by Logan et al. (1999).10
After ending the 45 min exposure, the concentration first decreased for ca.
15 min to a minimum of 0.3 ng ml−1, and then increased again for 120 min
up to 2.6 ng ml−1 before subsequently decreasing again. At the final sampling
point of these experiments, 195 min after ending the exposure, the concen-
tration in the blood was still as high as 0.9 ng ml−1. Toxicokinetic parameters
were not calculated from the measured concentration–time course, since no
meaningful fit could be obtained.
The observed pattern resembles that of the 8 min nose only exposure to a
Ct of 2400 mg min m−3. In Figure 7.4 the concentration–time profiles of sulfur
mustard in the blood during and after nose only exposure to a Ct of 2400 mg
min m−3 and percutaneous exposure to a Ct of 10 000 mg min m−3 are shown.
As for the inhalation toxicokinetics, the percutaneous toxicokinetics of sul-
fur mustard in the hairless guinea pig appear to be characterized by the exis-
tence of two absorption phases, one relatively rapid and one relatively slow.
The slow absorption phase may be due to absorption into the blood from
skin depots that were ‘loaded’ during the exposure. Just like for the inhala-
tion toxicokinetics, a ‘dip’ in the concentration in the blood is observed after
ending the exposure. This may be due to rapid distribution from the blood to
the tissues, as observed for the iv toxicokinetics. After ending the 8 min nose
only exposure, the animals had been exposed to a Ct of ca. 2400 mg min m−3.
At the 10 min time point of the percutaneous exposure, the animals had been
exposed to a Ct of 2500 mg min m−3. Upon comparison of the concentration–
time profiles in the blood for these two exposures, it is clear that the concen-
tration build-up of sulfur mustard in the blood proceeds more rapidly for
the nose only exposure, suggesting that absorption via the respiratory route
is more rapid than via the percutaneous route. Interestingly, the maximum
concentration in the blood observed for percutaneous exposure to 10 000 mg
min m−3 is approximately 3-fold higher than that for nose only exposure to
2400 mg min m−3. From the percutaneous toxicokinetic experiments it was
concluded that systemic intoxication from sulfur mustard is more likely to
206 Chapter 7
Figure 7.4 Mean

concentration–time courses (±standard error of the mean)
of sulfur mustard in the blood of anesthetized, restrained hairless
guinea pigs during and after 8 min nose only exposure to a Ct of 2400
mg min m−3 (•) and 45 min percutaneous exposure to a Ct of 10 000
mg min m−3 (o).
occur from a percutaneous exposure than from a respiratory exposure, at

least in the hairless guinea pig.
At just 10 min after ending the percutaneous exposure, considerable con-
centrations of intact sulfur mustard were measured in all tissues studied
(liver, lung, spleen, bone marrow, abdominal fat and dorsal skin). By far the
highest concentrations were observed in the skin, which is not surprising in
view of the high external load on the body of the animal. The tissue concen-
trations were generally higher than those observed after iv administration of
a dose corresponding to 0.3 LD50.
The DNA adduct levels in tissues were lower than anticipated on the basis
of the intact sulfur mustard concentrations. From most of the skin samples
DNA could not be isolated, indicating massive DNA damage had occurred,
leading to cross-linking between DNA and proteins.
7.4 T
he Influence of Scavengers on the
Toxicokinetics of Sulfur Mustard
For nerve agents, it has been demonstrated that the blood toxicokinetics
are influenced by administration of scavengers such as (human) butyrylcho-
linesterase, resulting in a lower AUC.6 The efficacy of potential scavengers
for sulfur mustard, i.e. N-acetylcysteine (NAC) and cysteine isopropyl ester
(CIPE), to protect against death from inhaled sulfur mustard was studied in
hairless guinea pigs by Langenberg et al. (1998).12 These compounds were
administered intraperitoneally 1 min prior to 5 min nose only exposure to
various concentrations of sulfur mustard vapor in air. From the mortality
rates at 96 h, the LCt50 values were calculated and compared with those in the
non-pretreated hairless guinea pig. Unfortunately, pretreatment with either
NAC or CIPE did not significantly increase the LCt50 value for respiratory
exposure to sulfur mustard. Consequently, it was not considered useful to
study the iv and inhalation toxicokinetics of sulfur mustard after administra-
tion of these compounds.
7.5 Toxicokinetics of Sulfur Mustard in Humans

Human volunteers have been exposed in a controlled way to sulfur mustard
liquid and vapor in order to observe the adverse effects on the exposed skin
(see overview by Papirmeister et al., 1991 16), whereas the effects on the respi-
ratory tract do not appear to have been studied. Furthermore, no reports can
be found that describe the toxicokinetics of sulfur mustard in humans.
There are, however, a few reports describing the effects of accidental expo-
sures of individuals to liquid sulfur mustard26,27 as well as the time course
of biomarkers for this exposure.28–30 Barr et al. (2008)28 followed protein bio-
markers in the plasma of two individuals who were exposed during destruc-
tion of ammunition dating from the First World War. They measured adducts
of sulfur mustard to cysteine-34 in albumin as well as adducts to glutamic
and aspartic acid in plasma proteins, of which the nature is not specified
further. One individual had chemical burns covering more than 6.5% of his
body surface area and was hospitalized. The other person only developed a
small blister. Both assays provided measurable concentrations of adducts in
the severely injured individual. Over a 40 day period, the concentrations of
the biomarkers decreased by ca. 75%. The half-life of disappearance of the
adduct to albumin was calculated to be ca. 21 days, which corresponds with
the half-life of human albumin, i.e. 17–23 days. The half-life of the adducts
to aspartic and glutamic acid residues in plasma proteins appeared to be a
little bit shorter (number not specified), which is tentatively explained by the
authors by the shorter half-life of the plasma proteins in question, or due to
hydrolytic or enzymatic release of the hydroxyethylthioethyl adduct from the
aspartic and/or glutamic acid residues. In the second patient the albumin
adduct could be detected in samples drawn in the first week after exposure.
Adducts to aspartic and glutamic acid residues could not be detected in this
patient.
Smith et al. (2008)29 reported the analysis of metabolites in the urine of
the same two patients. This included the free and conjugated simple hydro-
lysis products thiodiglycol (TDG) and its sulfoxide (TDG-sulfoxide), the
β-lyase metabolites 1,1′-sulfonylbis[2-(methylsulfinyl)ethane] (SBMSE) and
1,1′-methylsulfinyl-2-[2-(methylthio)-ethylsulfonyl]ethane (MSMTESE), as
208 Chapter 7
well as the precursor for the β-lyase metabolites 1,1′-sulfonylbis[2-(meth-
ylthio)ethane] (SBMTE). In the sample preparation, SBMSE and MSMTESE
were reduced to SBMTE by adding TiCl3. Consequently, all β-lyase metab-
olites were measured as SBMTE. In the severely exposed patient, TDG,
TDG-sulfoxide and SMBTE were detectable in the urine for up to 11 days
after the exposure. In the second patient low levels of SMBTE and TDG-
sulfoxide were measured in urine samples taken at days 2 and 7 after expo-
sure. It has to be realized that TDG and TDG-sulfoxide cannot be consid-
ered specific biomarkers for sulfur mustard exposure, since background
levels are present in the urine of individuals who have definitely not been
exposed to this agent. The authors conclude that the β-lyase metabolites
are the best urinary biomarkers for sulfur mustard exposure. The half-life
of these specific biomarkers in the urine was calculated to be less than 1
day in these two patients.
Recently, Xu et al. (2014)30 reported a case of four individuals exposed to
an unknown viscous oily liquid from a barrel at a construction site, followed
by blister formation and hospitalization. In view of the signs and symptoms,
exposure to sulfur mustard was suspected, since many abandoned chemical
warfare agents are still present in China. Blood, urine and exudate samples
were collected from the victims and analyzed for four types of biomarkers
associated with sulfur mustard exposure: (i) hydrolysis products (TDG and
TDG-sulfoxide); (ii) β-lyase metabolites [SBMTE, MSMTESE, SMBSE and
1,1′-sulfonylbis[2-S-(N-acetylcysteinyl)ethane] (SBSNAE)]; (iii) DNA adducts
to guanine (N7 and O6) and adenine (N3) as well as bis[2-(guanine-7-yl)ethyl]
sulfide (Bis-G); and (iv) hemoglobin adducts, mainly to the N-terminal valine.
The concentrations of TDG and its sulfoxide in urine were higher than the
background value on day 3 in three out of four patients. The concentration
of the hydrolysis/oxidation products in the blister exudate were considered
to be in good accordance with the severity of the injury. The oxidation prod-
uct of sulfur mustard, bis-β-chloroethyl sulfoxide, was not detected in the
urine, but was detectable at low levels in the plasma of the patients, as well
as in the blister exudate. β-Lyase metabolites were detected in all samples
drawn from the patients, with the lowest concentration in the most severely
intoxicated patient. The authors hypothesize that this may be due to a rapid
depletion of glutathione in this patient. The four DNA adducts were detected
in the urine up to 32 days after exposure, with maximum values on days 4–7.
The most abundant adduct was the N7-guanine adduct. In the more severely
intoxicated patients, the adduct concentrations in urine were higher than in
the patient with a less severe injury. The four DNA adducts were detectable
in the blood until day 14. The adduct to the N-terminal valine of hemoglobin
was found in the blood of the patients at high concentrations on days 3–7,
with levels declining from the second week after exposure, but still detect-
able in the blood of one of the patients 3 months after exposure. The authors
conclude that this set of biomarkers is of great importance for early diagno-
sis of exposure to sulfur mustard and for monitoring the treatment of intox-
ications by this agent.
The reported findings are in reasonable agreement with those of Benschop
et al. (1997),31 who detected the N7-adduct to guanine and the N-terminal
valine adduct of hemoglobin in the blood of two Iranian victims 22–26 days
after the alleged exposure. One of the victims showed clear signs of sulfur
mustard exposure, i.e. skin injuries but no respiratory effects, whereas the
other victim showed symptoms that were only vaguely compatible with sul-
fur mustard.
7.6 Conclusions
The toxicokinetics of sulfur mustard have been studied in various species
for several exposure routes, including the militarily relevant respiratory and
percutaneous routes. Although all studied animal models have produced
valuable data, there are some marked interspecies differences. For the per-
cutaneous route, the hairless guinea pig appeared to be a convenient and
suitable small animal model. Unfortunately, this model is only available at
high cost and in very limited numbers. The skin of the pig is quite compa-
rable to that of humans, but due to its size it is a less convenient animal
for toxicokinetic studies involving controlled vapor exposures. Meanwhile,
the mini-pig is becoming more popular as an animal model, and might be
a suitable alternative to the hairless guinea pig and normal pig. However,
this animal model is more demanding in terms of facilities than rodents and
guinea pigs. For the respiratory route, the marmoset monkey is clearly the
better model for humans. The model is expensive and the use of non-hu-
man primates is under increasing societal scrutiny. Although the anatomy
of the upper airways of the mini-pig is quite different from that of humans,
the mini-pig could be a suitable alternative for the non-human primate, for
instance by circumventing nasal absorption.
In a sulfur mustard vapor environment, unprotected military personnel
will be exposed simultaneously via the respiratory and percutaneous routes.
Such a combined exposure has not yet been studied, but it would be interest-
ing to see which route would be predominant. Obviously, the environmental
conditions will have a profound influence on the relative importance of the
two exposure routes.
The generated toxicokinetic data can be used for validation of physiolog-
ically based toxicokinetic models. Such models should take into account
that sulfur mustard may influence physiological parameters in a dose
dependent way.
From the findings reported in this chapter, it can be concluded that despite
the high reactivity of sulfur mustard the compound is remarkably stable in
vivo, which is in line with the observations of Drasch et al. (1987),32 who mea-
sured intact sulfur mustard at the micrograms per gram level in an Iranian
soldier who died from pneumonia 7 days after a severe exposure. In view of
the high persistence of sulfur mustard in the body, a scavenger approach
should be feasible. The cysteine derivatives studied so far appeared not to
provide any protection. Therefore, other compounds with a high affinity for
210 Chapter 7
sulfur mustard and/or its reactive intermediates need to be identified and
tested. Concomitantly, research is still needed to elucidate the mechanism of
action of sulfur mustard in order to find clues for a causally based therapy of
intoxication by this agent. However, sulfur mustard research does not appear
be an area of high priority at present, and thus it seems unlikely that a break-
through will be accomplished in the coming years.
References
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H. P. M. van Helden, Toxicokinetics of the four stereoisomers of the nerve
agent soman in atropinized rats – Influence of a soman simulator, Toxi-
col. Appl. Pharmacol., 1987, 90, 490.
2. I. Koplovitz, L. W. Harris, D. R. Anderson, W. J. Lennox and J. R. Stewart,
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3. J. J. Gordon, L. Leadbeater and M. P. Maidment, Protection of animals
against organophosphate poisoning by pretreatment with a carbamate,
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4. M. J. van der Schans, H. J. Lander, H. J. van der Wiel, J. P. Langenberg and
H. P. Benschop, Toxicokinetics of the nerve agent (±)-VX in anesthetized
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7. H. John, F. Balszuweit, K. Kehe, F. Worek and H. Thiermann, Toxicoki-
netic Aspects of Nerve Agents and Vesicants, in Handbook of Toxicology
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abrasion–a novel concept in the surgical management of sulphur mus-
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mosets. Efficacy of Nasal Scavengers, Final report for USAMRMC contract
DAMD17-03-1-0613, ADA441282, 2005.
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Mars-Groenendijk, F. J. Bikker, G. P. van der Schans, H. P. Benschop and
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set, Proceedings NATO TG-004, Hradec Kralove, CZ, 2005.
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H. P. M. van Helden, G. P. van der Schans and H. P. Benschop, Toxicoki-
netics of sulfur mustard and its DNA-adducts in the hairless guinea pig,
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and nitrogen mustards, in Chemical warfare agents and related chem-
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212 Chapter 7
Development, National Defense Research Committee, 1946, vol. 4,
ch. 23, pp. 479–518.
25. M. Bergmann, J. S. Fruton, C. Golumbic, S. M. Nagy, M. A. Stahmann and
W. H. Stein, The penetration of vesicant vapors into human skin, OSRD 4855,
The Rockefeller Institute for Medical Research, 1945.
26. H. Q. Le and S. J. Knudsen, Exposure to a First World War blistering
agent, Emerg. Med. J., 2006, 23, 296.
27. J. Newmark, J. M. Langer, B. Capacio, J. Barr and R. G. McIntosh, Liquid
sulfur mustard exposure, Mil. Med., 2007, 172, 196.
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Solano, J. V. Wooten, S. W. Lemire, J. D. Thomas, D. H. Ash and D. L. Ash-
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29. J. R. Smith, B. R. Capacio, W. D. Korte, A. R. Woolfitt and J. R. Barr, Analysis
for plasma protein biomarkers following an accidental human exposure
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Lin, B. Wu, J. Feng, H. Li, L. Guo and J. Xie, Four sulfur mustard exposure
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31. H. P. Benschop, G. P. van der Schans, D. Noort, A. Fidder, R. H. Mars and
L. P. A. De Jong, Verification of exposure to sulfur mustard in two casualties
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32. G. Drasch, E. Kretschmer, G. Kauert and L. von Meyer, Concentrations
of mustard gas [bis(2-chloroethyl)sulfide] in the tissues of a victim of a
vesicant exposure, J. Forensic Sci., 1987, 32, 1788.
Chapter 8
Modeling Organophosphorus
Chemical Warfare Nerve
Agents: A Physiologically
Based Pharmacokinetic–
Pharmacodynamic (PBPK-PD)
Model of VX
Tammie R. Covington*a, Lucille A. Lumleyb,
Christopher D. Ruarka, Edward D. Clarksonb,
Christopher E. Whalley c, and Jeffery M. Gearhart a
a
Henry M. Jackson Foundation for the Advancement of Military
Medicine, 711 HPW/RHDJ, Wright-Patterson AFB, OH, USA; bUS Army
Medical Research Institute of Chemical Defense, Aberdeen Proving Ground,
MD, USA; cUS Army Edgewood Chemical Biological Center, Aberdeen
Proving Ground, MD, USA
*E-mail: tammie.covington.ctr@us.af.mil

213
214 Chapter 8
8.1 Introduction
Chemical warfare nerve agents pose a potential threat to the general pub-
lic as well as the military, as evidenced by several incidents. Between 1980
and 1988, sarin (GB) was used by Iraq in the war with Iran, with the most
notable incident occurring in 1988 when a Kurdish city in northern Iraq was
bombarded with chemicals, possibly including GB, tabun (GA) and O-ethyl
S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX).1 In 1994 and
1995, the Aum Shinrikyo sect attacked subways in Matsumoto and Tokyo
with GB, and also attacked individuals with VX in Osaka and Tokyo. One of
these individual attacks resulted in the death of the intended victim. The vic-
tim had VX deposited on his neck and exhibited symptoms typical of organo-
phosphate poisoning, but confirmation of the nerve agent used could only
be achieved after his death with the testimony from one of the suspected
attackers and detection of VX metabolites [ethyl methylphosphonic acid
(EMPA) and 2-(diisopropylamino-ethyl)methyl sulfide (DAEMS)] in a blood
sample taken approximately 1 h after the attack.
Chemical warfare nerve agents are highly toxic chemicals that act by inhib-
iting acetylcholinesterase (AChE), resulting in an accumulation of acetylcho-
line. This accumulation can result in severe muscle contractions, paralysis
and possibly death if untreated. VX is one of the most potent nerve agents,
with an estimated percutaneous 50% lethal dose (LD50) for humans that is
approximately 200 times smaller than the percutaneous LD50 for GB (10 mg
versus 1700 mg) and an estimated inhalation 50% lethal concentration
(LCt50) that is about half that for GB (30 mg min m−3 versus 70 mg min m−3).2
Typical treatment of nerve agent exposure includes administration of atro-
pine, oximes such as pralidoxime (2-PAM), and diazepam. Unfortunately,
the countermeasures that are most effective for nerve agents such as GB and
soman (GD) are less effective against VX, possibly due to the longer clearance
time of VX from the body.
In order to improve risk estimates for VX exposures, the pharmacodynam-
ics and, consequently, the pharmacokinetics must be better understood;
physiologically based pharmacokinetic–pharmacodynamic (PBPK-PD) mod-
els can be useful analytical tools for this purpose. A physiologically based
pharmacokinetic (PBPK) model is a mathematical description, typically
using ordinary differential equations, that describes the absorption, distri-
bution, metabolism and excretion of a chemical by the body. It allows for
the coordination of species specific physiology, chemical specific character-
istics, and the experimental protocol for the chemical and exposures of con-
cern. PBPK-PD models incorporate descriptions of the pharmacodynamics
into the pharmacokinetic descriptions, allowing for the prediction of the
response to a given internal concentration. These models allow for the deter-
mination of better estimates of the actual chemical dose delivered to a target
tissue as well as the potential response resulting from this dose as opposed
to estimating a response based on the external dose alone.3,4 The physiologi-
cal nature of these models also allows for extrapolation between dose routes
Modeling Organophosphorus Chemical Warfare Nerve Agents 215
as well as between species, and for simulation of simultaneous exposures via
multiple routes (e.g., concurrent inhalation and dermal exposure).
Relatively few PBPK models have been developed to describe the pharmaco-
kinetics and pharmacodynamics of chemical warfare nerve agents. Maxwell
et al.5 developed a PBPK-PD model for GD in the rat, describing the inhi-
bition of AChE and carboxylesterase (CaE) in blood and tissues with mass
balance equations based on parameters for blood flow, tissue volumes, GD
metabolism and tissue/plasma partition coefficients. The resulting model
gives accurate predictions of AChE activity in the blood and seven different
tissues following intramuscular dosing with 90 µg GD kg−1 bodyweight (BW),
and was able to reproduce dose–response AChE inhibition from 10 to 100%
in the brain.
Gearhart et al.6,7 developed a PBPK-PD model for diisopropylfluorophos-
phate (DFP) as a surrogate compound for other highly toxic organophos-
phates, such as chemical warfare nerve agents, to describe pharmacokinetics
and pharmacodynamics in the blood and various tissues. This model con-
struct was able to predict the pharmacokinetics of DFP and inhibition of
AChE and butyrylcholinesterase (BuChE) after acute intravenous dosing in
mice, repeated subcutaneous dosing in rats, and single and repeated intra-
muscular dosing in humans. Predictions were then generated with the model
for brain DFP concentrations in humans following an inhalation exposure.
This DFP model was then modified to create a PBPK model for GB4 that
includes tissue compartments for the eye and skin. The model was also mod-
ified to account for scrubbing in the upper airways. The GB model was used
to predict guinea pig data for the blood GB concentration following intrave-
nous dosing; blood AChE activity and red blood cell (RBC) regenerated GB
following subcutaneous dosing; and RBC regenerated GB following inhala-
tion exposure. The model was also used for some cross-route comparisons.
This chapter describes the development of a PBPK-PD model for VX expo-
sure based on these modeling efforts with the goal of developing a multispe-
cies model that may then be used to simulate exposure in humans in order
to improve the current risk estimates for VX exposures. To date, a PBPK-PD
model for VX has not been published.
8.2 Materials and Methods

8.2.1 PBPK Model Structure
The PBPK-PD model presented here is based on previously developed
PBPK-PD models4,6,7 describing the kinetics and dynamics resulting from
DFP or GB exposure, and has diffusion limited compartments for the brain,
diaphragm, fat, kidneys, liver, lungs, skin, and the remaining rapidly and
slowly perfused tissues (Figures 8.1 and 8.2). The model includes B-esterase
(AChE, BuChE and CaE) activity (Figure 8.3) in all tissues except fat;8 metab-
olism of VX via Michaelis–Menten kinetics in the blood, brain, kidneys, liver
and rapidly perfused compartment;4,6 and first-order urinary excretion from
216 Chapter 8

of the PBPK-PD model for VX. Note that the compartments
denoted by dashed boxes only exist when dosing via that route. SC:
subcutaneous.

of the coding for each tissue in the PBPK-PD model for VX.
Arterial and venous blood are also coded to have B-esterase inhibition;
fat is not coded to have B-esterase inhibition.

of the sub-model for B-esterase inhibition.
the kidney. Metabolism in the skin and non-B-esterase binding in the blood,
through a set of terms that represent both rapid binding and a more delayed
binding, are also included as indicated when fitting the data of Lumley et al.9
and van der Schans et al.10 The resulting system of equations is solved using
the Gear algorithm in acslX (AEgis Technologies Group, Inc., Huntsville, AL).
8.2.1.1 Routes
The model is capable of simulating exposure from three routes: intravenous,
subcutaneous and dermal. Although the intravenous route may not be a very
probable real world exposure route, data from intravenous dosing allow for
the estimation of unknown parameters without the uncertainty of route
specific kinetics such as permeability or absorption. Intravenous exposures
are modeled as injections directly into the venous blood supply over a short
period of time.
Simulation of the subcutaneous route acts as a surrogate for dermal dos-
ing without the uncertainties in actual exposure area, skin thickness or
permeability at the dosing site. Subcutaneous dosing is simulated with a
separate compartment that is subtracted from the slowly perfused compart-
ment, allowing the injected dose to be treated as a concentrated amount at
the injection site rather than diluted across the volume of the entire slowly
perfused compartment. This subcutaneous dosing compartment is created
within the model code only when a subcutaneous dose is being given and
does not exist when dosing via another route. Subcutaneous dosing is mod-
eled as an injection directly into the subcutaneous dosing compartment at a
constant rate with uptake into the blood pool through perfusion of the sub-
cutaneous dosing compartment.
Given VX’s low volatility, simulation of the dermal route is important
as it is the most likely real world route of exposure. Dermal absorption is
described using Fick’s law11,12 and uses a separate dosing compartment that
is subtracted from the skin compartment. It is only created within the model
code when a dermal dose is being given and does not exist when dosing via
another route. Evaporation from the dermal exposure site is considered to
be negligible given the low volatility of VX13 and is thus not included in the
model.
218 Chapter 8
8.2.1.2 Endpoints
The current model can simulate various endpoints for blood and tissues
including free VX concentrations and B-esterase activity (AChE, BuChE and
CaE). The model also has the capability to adjust cardiac output and tissue
blood flows based on the level of brain AChE activity in order to mimic the
physiological response to AChE inhibition. This adjustment assumes that
cardiac output and tissue blood flows decrease over time by the same frac-
tion as brain AChE activity. This adjustment is made when simulating data
where the animals were not given atropine and were not artificially venti-
lated; otherwise, the adjustment was not made.
8.2.2 Model Parameters

Parameters were obtained from the published literature whenever pos-
sible; those not available from the literature were either estimated using
quantitative structure–activity relationship (QSAR) techniques or fit using
the model and available data. All simulations presented in this paper use
the same set of parameters (Tables 8.1–8.4) with the exception of route
or study specific parameters (e.g., dermal exposure area, body weight).
In order to simplify intra- and inter-species extrapolation, allometric
scaling is used throughout the model with volumes scaled by BW and
blood flows, and metabolic capacities scaled by BW to the three-quarter
power;14,15 however, the current model is parameterized only for the phys-
iology of guinea pigs.
8.2.2.1 Physiological Parameters
All simulations use study specific BWs when available. Physiological param-
eter values used and their corresponding reference are presented in Table
8.1. Values taken from Peeters et al.16 are for a non-pregnant guinea pig.
The value for fractional blood flow to fat17 is for rat since a guinea pig value
could not be located. Arterial and venous blood volumes are calculated
using the total blood volume18 and an arterial/venous blood split of 30/70
based on the arterial and venous values in Sweeney et al.19 Fractional blood
flow and volume for the rapidly perfused compartment represent the entire
rapidly perfused tissue pool, and values for the brain, kidneys and liver are
subtracted from the total rapidly perfused values in the model for simu-
lating the rapidly perfused compartment. The same is true for the slowly
perfused compartment with values for the diaphragm, fat and skin being
subtracted from the total slowly perfused values. The blood flow to the sub-
cutaneous and dermal dosing compartments are computed as a portion of
the flow to the slowly perfused or skin compartments, respectively, based
on the ratio of the volumes of the respective dosing and tissue compart-
ments. The volume of the dermal dosing compartment is defined based on
Table 8.1 Physiological
parameters.
Fractional Fractional tissue Fractional tissue
Parameter blood flow volumea blood volume Other
Tissue parametersb
Arterial blood — 0.0178f,g — —
Brain 0.0198c 0.00491c 0.0198h —
Diaphragm 0.003d 0.003d 0.0379h —
Fat 0.07e 0.014f 0.0154h —
c
Kidneys 0.145 0.00982c 0.1984h —
Liver 0.18f 0.0424c 0.1488h —
Lungs — 0.0113c 0.2069h —
Rapidly perfused 0.73f 0.11f 0.122h —
compartment
Skin 0.117c 0.186c 0.02e —
Slowly perfused 0.27f 0.82f 0.026h —
compartment
Venous blood — 0.0416f,g — —
Dermal dosing Calculated in model 0.02i —
compartment
Subcutane- Calculated in model 0.026j —
ous dosing
compartment

Other parameters
Cardiac output — — — 16.157c
(l h−1 kg−0.75)
Hematocrit — — — 0.42h
(unitless)
Skin thickness — — — 0.0075k,l
(cm)
a
Includes blood.
b
Blood flows are fractions of cardiac output, volumes are fractions of body weight and blood
volumes are fractions of tissue volume.
c
Peeters et al.16—non-pregnant guinea pig value.
d
Langenberg et al.57
e
Brown et al.17—rat value.
f
Sweeney et al.19
g
Langenberg.18
h
Bosse and Wassermann.20
i
Used skin value.
j
Used slowly perfused value.
k
Ferry et al.21
l
Middelkamp-Hup et al.22
the area exposed and skin thickness. The brain blood volume is calculated
using a brain plasma volume and hematocrit value.20 Values for fractional
blood volume for the rapidly and slowly perfused compartments are esti-
mated by averaging values from Bosse and Wassermann20 that would fall
into each of the two compartments. A rat value17 is used for the skin blood
volume. The value for skin thickness is the sum of values for the stratum
corneum21 and epidermal thickness.22
Table 8.2 Chemical
specific parameters.
220
Tissue/blood Diffusion limitation Maximum metabolic Michaelis–Menten
Parameter partition (unitless) (l h−1 kg−0.75) rate (µg h−1 kg−0.75) affinity (µg l−1) Other
Partition coefficients, diffusion constants and metabolism parameters
Blood — — 2500.0d 2500.0d —
Brain 3.82a 1.0d 0.1d 0.1d —
a,b
Diaphragm 1.35 0.0007d — — —
Fat 1.54a 0.00073f — — —
a
Kidneys 2.40 0.08d 1000.0d 1000.0d —
Liver 2.25a 0.08g 10 000.0d 10 000.0d —
a
Lungs 0.93 0.006d — — —
Rapidly perfused compartment 2.25a,c 0.08g 100.0d 100.0d —
a
Skin 1.21 0.0007f 1.5d 100.0d —
Slowly perfused compartment 1.35a,b 0.0007f — — —
d
Dermal dosing compartment 800.0 0.0007f 1.5h 100.0h —
Subcutaneous dosing compartment 0.0025/0.04/0.25e 0.0007f — — —
Skin/saline partition 1.0d — — — —

Other parameters
Metabolite clearance (l h−1 kg−0.75) — — — — 0.01d
Volume of distribution for metabolite — — — — 0.8d
(fraction of BW)
Urinary excretion rate (l h−1 kg−0.75) — 0.005d
Non-B-esterase fast on binding rate (h−1) — — — — 90.0d
Non-B-esterase fast off binding rate (h−1) — — — — 5.0d
Non-B-esterase slow on binding rate (h−1) — — — — 20.0d
Non-B-esterase slow off binding rate (h−1) — — — — 0.02d
a
Predicted using quantitative structure–activity relationship (QSAR) techniques.23
b
Used muscle value.
c
Used liver value.
Chapter 8
d
Fit to data.
e
Values for 0.8, 3.2 and 8 µg kg−1 doses, respectively (see text for further details).
f
Used diaphragm value.
g
Used kidney value.
h
Used skin value.
Table 8.3 Pharmacodynamic
parameters.
Parameter AChE BuChE CaE
−1
Initial B-esterase concentrations of binding sites (µm l )
Blood 0.0097a 0.0423j 0.44b
a
Brain 0.12 0.0117j 0.549m
Diaphragm 0.0023b 0.0046k 0.533m
a
Kidneys 0.0044 0.0119j 11.0b
Liver 0.0063a 0.0848j 65.9b
a
Lungs 0.0045 0.0193j 0.275b
Rapidly perfused compartment 0.0491b 0.0165k 0.46b
a,c
Skin 0.0016 0.002k 0.33b,e
Slowly perfused compartment 0.0016a,c 0.0029k 0.33b
Dermal dosing compartment 0.0016d 0.002d 0.33d
Subcutaneous dosing compartment 0.0016e 0.0029e 0.33e

Rate constants
Bimolecular inhibition (µm−1 l−1 h−1) 2000.0f 230.0f 0.09n
Enzyme reactivation (h−1) 0.021g 0.15f 0.021o
Enzyme aging (h−1) 0.014h 0.009l 0.0p
Enzyme degradation (h−1) 0.0042i 0.022i 0.0042o
a
angenberg et al.57
L
b
Sweeney et al.19
c
Used muscle value.
d
Used skin value.
e
Used slowly perfused value.
f
Fit to data.
g
Worek et al.58—human value.
h
Maxwell et al.53
i
Estimated (see text).
j
Langenberg.18
k
Sweeney and Maxwell.59
l
Worek et al.55—human value.
m
Chen and Seng.60
n
Maxwell34—rat value.
o
Used AChE value.
p
Maxwell and Brecht24—rat value.
Table 8.4 Dosing

parameters.
Parameter Intravenous Subcutaneous Dermal
a
Length of injection (h) 0.001667 0.001667a —
Volume of dose (l kg−1) — 0.0005b —
Multiple for dose volume (unitless) — 2.0a —
Initial concentration on skin (µg l−1) — — Study specificc
Exposure area (cm2) — — Scaled by dosed
Permeability constant (cm h−1) — — 0.001e
a
Estimated.
b
Specified in study protocol.9,29–31
c
Calculated from dose, BW and volume applied for Lumley et al.9 data and from the dilution
rate for Benschop et al.27 and van der Schans et al.10 data.
d
Calculated by scaling from measured spread for Lumley et al.9 dose to larger dose by amount
of solution applied.
e
Fit to data.
222 Chapter 8
8.2.2.2 Chemical Specific Parameters

Chemical specific parameters are summarized in Table 8.2. Rat estimates for
partition coefficients23 are used due to the lack of all necessary guinea pig
data for input into the algorithm. The partition coefficient for the dermal
dosing compartment is fit to the data and varies from the partition used for
the skin compartment to account for the fact that the dose of VX was diluted
in isopropanol. Partition coefficients for the subcutaneous dosing compart-
ment for various dilution rates are also fit to the data. Diffusion limitation
constants are fit to the data or are set to either the diaphragm value or kid-
ney value due to the lack of data for these tissues. The remaining chemical
specific parameters are fit to the data. Due to the paucity of data to fit the
metabolic parameters for each of the tissues, it was initially assumed that
the fraction of liver metabolism in each non-liver tissue would be the same
as in the DFP model.6 Metabolism parameters for the brain and skin were
subsequently changed in order to better fit the data.
8.2.2.3 Pharmacodynamic Parameters
Specific values and corresponding references are presented in Table 8.3. The
reactivation rate for AChE is used for CaE due to the lack of data. The aging
rate for BuChE is calculated from the half-life; the rate for CaE is assumed to
be zero based on information provided for the rat in Maxwell and Brecht.24
Degradation rates for AChE and BuChE are based on the RBC lifespan of 30
days for guinea pigs25 and fitting of the model to data on AChE and BuChE
recovery following inhalation exposure to GB.26 Due to a lack of information,
the value for AChE is used for CaE. Synthesis rates for AChE, BuChE and CaE
are calculated within the model based on the degradation rates such that a
steady-state B-esterase level is maintained in the absence of a dose. These
parameters are also presented in Table 8.3.
8.2.2.4 Dosing Parameters
Dosing parameters are given in Table 8.4. The volume of the subcutaneous
dosing compartment is defined arbitrarily as twice the volume of the injected
dose; in conducting the sensitivity analyses (see Sections 8.3 and 8.4), it is
shown that the selected endpoints are not more sensitive to this parameter
than they are to any other dosing parameter.
8.2.3 Data Used for Model Parameterization and Validation

Fit parameters are based on data for all three routes; these parameter values are
then used for the final simulations of these data as well as the simulations for
the validation data from these and additional studies. Some of the data used are
from Benschop et al.,27 which is a more inclusive technical report of the same
study published by van der Schans et al.;10 however, it is a limited distribution
document so data reported solely in this report are not presented here.
8.2.3.1 Intravenous Data
The model is parameterized using the pharmacokinetic data of Benschop
et al.27 and van der Schans et al.10 and is validated with the pharmacokinetic
data of Trap.28 All of the animals in the Benschop et al.27 and van der Schans
et al.10 study received atropine and were artificially ventilated during dosing.
The Trap28 report is not in English, so it could not be determined whether
animals received atropine or artificial ventilation during dosing. Since this
study and the Benschop et al.27 and van der Schans et al.10 studies appear
to have been conducted at TNO in The Netherlands, it is assumed that this
study was conducted using a similar protocol.
8.2.3.2 Subcutaneous Data
Pharmacodynamic data of Lumley et al.9 are used to parameterize the model,
which is then validated with pharmacodynamic data from Shih et al.29–31 Only
the animals in the 2011 and 2005 studies29,31 were given atropine prior to the
subcutaneous injection of VX and none of the animals given subcutaneous
doses were artificially ventilated during exposure.
8.2.3.3 Dermal Data
The model is parameterized using pharmacokinetic and pharmacodynamic
data from Lumley et al.9 and is validated with pharmacokinetic and phar-
macodynamic data from Benschop et al.27 and van der Schans et al.10 The
animals in the Lumley et al.9 study received neither atropine nor artificial
ventilation during exposure, but the Benschop et al.27 and van der Schans et
al.10 animals received both. The area of exposure to use for simulating the
validation dataset is estimated using body weight, dose and dilution rate for
the validation dataset and the spread observed for the 60 µg VX kg−1 BW dose.
8.2.4 Sensitivity Analyses

Sensitivity analyses were conducted to determine the impact of variations
in input parameters upon selected output parameters. Input parameters
were varied by 1% and the resultant sensitivity coefficients were determined.
Coefficients presented here are normalized to both the response variable
and the parameter being varied and, thus, are defined as fractional change
in response per fractional change in input. A negative coefficient indicates
a decrease in the response variable with an increase in the input parameter,
and a coefficient greater than 1 in absolute value indicates a larger than one
to one relationship between the response and input.
A sensitivity analysis is conducted for each of the three dose routes by simu-
lating guinea pig exposure to a single dose of either 28 µg VX kg−1 BW intrave-
nously, 3.2 µg VX kg−1 BW subcutaneously, or 60 µg VX kg−1 BW dermally. These
analyses are all conducted by adjusting the appropriate parameters (i.e., only
224 Chapter 8
those parameters valid for a given dose route) from Tables 8.1–8.4 by 1%. Expo-
sure is simulated for 10 h for the intravenous dose and for a longer period of
50 h for the subcutaneous and dermal doses due to the longer uptake time for
these two routes. These analyses are run both with and without adjusting car-
diac output and tissue blood flows based on brain AChE activity to determine
the impact of this adjustment in conjunction with the individual parameters.
Selected endpoints for these analyses are blood VX concentration and AChE
activity in the blood, brain and diaphragm. In addition to blood AChE activity,
activity levels in the brain and diaphragm are included to cover targets of both
the central nervous system and potential peripheral target sites.
8.3 Results
8.3.1 Simulations
Results of the model simulations are show in Figures 8.4–8.11. Overall,
simulations are quite good and are within 2 standard deviations of almost
all data points and within 1 standard deviation of most data points. It is
worth noting that, while some of the data may not appear to be as well
simulated, at some time points some data for B-esterase activity in tis-
sue are actually measured with fraction of control values above 1, and
the model was not coded to account for concentrations above baseline or
control values.
8.3.1.1 Data for Fitting

Figure 8.4 shows the simulations of the intravenous (28 and 56 µg VX kg−1
BW) data of van der Schans et al.,10 while simulations of the subcutaneous
(3.2 µg VX kg−1 BW) and dermal (60 µg VX kg−1 BW) data of Lumley et al.9
are shown in Figures 8.5–8.7. In order to accurately simulate the tri-pha-
sic nature of the blood concentration data following intravenous dosing,
both rapid and slow non-B-esterase binding are incorporated into the
model. Parameters fit using intravenous blood concentration data and
metabolite data from Benschop et al.27 are those for metabolism, B-es-
terase pharmacodynamic parameters and non-B-esterase binding. Metab-
olism and B-esterase pharmacodynamic parameters have been fitted,
along with parameters for diffusion, based on the subcutaneous dosing
data. Dermal data are also used to fit parameters for B-esterase kinetics
and to fit the skin diffusion parameter, the skin/saline partition and the
permeability parameter.
8.3.1.2 Data for Validation

Simulations for the intravenous validation data28 are shown in Figure 8.8,
and simulations for the subcutaneous datasets (0.8 µg VX kg−1 BW9 and 8 µg
VX kg−1 BW29–31) are shown in Figures 8.9 and 8.10, respectively. Figure 8.11
Figure 8.4 Simulation

of blood VX concentration (ng ml−1) of male hairless guinea
pigs following a single intravenous dose of 28 µg VX kg−1 BW (solid line)
or 56 µg VX kg−1 BW (dashed line) (1 or 2 LD50). Symbols represent mea-
sured means ±2 standard errors of the mean10 for doses of 28 µg VX kg−1
BW (triangles) or 56 µg VX kg−1 BW (squares). Inset shows simulation
and data for early time points.
shows simulations for the validation dataset for dermal dosing with 125 µg
VX kg−1 BW.10 The intravenous simulations are good and might be better if
the experimental details had been better determined. For the 0.8 µg VX kg−1
BW subcutaneous data, the general shapes of the simulations agree with
the data, although AChE activity in the blood is consistently under-pre-
dicted (i.e., over-prediction of inhibition). The data for brain AChE activity
following the 8 µg VX kg−1 BW subcutaneous dose are for specific brain
regions whereas the model has only a general brain compartment; there-
fore, the model simulation is plotted against all of the brain region data
rather than one particular region or the average of the data points for the
regions. The dermal validation simulations are quite good, particularly con-
sidering that there are some strain and dosing site differences between the
dermal experiments used for fitting and validation as well as differences in
dilution rates, and the only parameter changes made between the two sim-
ulations were those that are experiment specific (BW, dose, exposure area
and initial concentration on skin).

Tables 8.5–8.7 present the most extreme point for the time course of the
sensitivity coefficients (i.e., the largest coefficient in absolute value) for
both adjusting and not adjusting cardiac output and tissue blood flows for
brain AChE activity. These values are used to categorize the model’s sen-
sitivity to parameters as low, medium or high, with low sensitivity being
defined as coefficients less than 0.2 in absolute value, medium sensitivity
as greater than or equal to 0.2 and less than or equal to 0.5 in absolute
226 Chapter 8

of (a) AChE and (b) BuChE activity, as a fraction of baseline
measurements, in whole blood of male Duncan Hartley guinea pigs fol-
lowing a single subcutaneous dose (line) of 3.2 µg VX kg−1 BW (0.4 LD50).
Triangles represent measured means ±2 standard errors of the mean.9
Inset shows simulation and data for early time points.
value, and high sensitivity as greater than 0.5 in absolute value.32 Results
for parameters for which the model has low sensitivity are not presented
in either the tables or figures. Time courses of the sensitivity coefficients
for simulations when adjustments are made based on brain AChE activity
are also presented in Figures 8.12–8.23 to demonstrate both the variabil-
ity in sensitivity across time and the time periods for which the model is
most sensitive. Time courses for the maximum metabolism rate for blood
are mirror images of the time courses for the corresponding Michaelis–
Menten affinities and are thus not shown. Figures for simulations when
adjustments are not made are also not shown. Some of the figures do not
show early time points as there were no changes in the sensitivity coeffi-
cients for these time points.

of AChE activity, as a fraction of control measurements, in
tissues of male Duncan Hartley guinea pigs following a single subcuta-
neous dose (solid line) of 3.2 µg VX kg−1 BW (0.4 LD50) or a single dermal
dose (dashed line) of 60 µg VX kg−1 BW (0.4 LD50). Symbols represent
measured means ±2 standard errors of the mean9 for the subcutaneous
(triangle) and dermal (squares) routes.
8.3.2.1 Sensitivity Analyses for the Intravenous Route

The endpoints for the intravenous route are sensitive to five of the parameters
(Table 8.5) only if cardiac output and tissue blood flows are adjusted for brain
AChE activity (see note c of Table 8.5). In general, sensitivities are highest at
the times when the endpoint of concern is most rapidly changing or is very
close to zero. With blood VX concentration, these are the time periods early
during the injection period as blood levels are rising and late in the simulation
as blood levels become quite low (Figure 8.12). For AChE activity in the blood,
brain and diaphragm (Figures 8.13–8.15), these time periods are just after the
injection period, as activity levels are rapidly changing or are at very low levels,
with the increased delay between the sensitivities for the brain and diaphragm
versus blood activity reflecting the delay in time for VX to reach the brain and
diaphragm. Thus, the larger number of parameters to which the endpoints
of brain and diaphragm AChE activity are sensitive is not completely unex-
pected as there are numerous parameters that affect how quickly and how
much VX reaches the brain and diaphragm, as well as the availability of VX
once it reaches the tissue. Also for brain and diaphragm activity, more param-
eters seem to exhibit the same pattern of sensitivity coefficients, whereas the
228 Chapter 8

of (a) AChE and (b) BuChE activity, as a fraction of base-
line measurements, in whole blood, and (c) amount of VX on the skin
surface for male Duncan Hartley guinea pigs following a single dermal
dose (line) of 60 µg VX kg−1 BW (0.4 LD50). Triangles represent measured
means ±2 standard errors of the mean.9 Inset shows simulation and
data for early time points.

of blood VX concentration (ng ml−1) of guinea pigs follow-
ing a single intravenous dose of 9 µg VX kg−1 BW (solid line), 18 µg VX
kg−1 BW (dashed line) or 54 µg VX kg−1 BW (dash-dot-dot line). Symbols
represent measured means28 for doses of 9 µg VX kg−1 BW (triangles),
18 µg VX kg−1 BW (squares) and 54 µg VX kg−1 BW (circles). Inset shows
simulation and data for early time points.
patterns are more varied for the other two endpoints. Increased sensitivities
during the early and later time periods could partly be a result of numerical
imprecision, as opposed to just model sensitivity, due to the rapid changes
in endpoint values at the earlier time points and very small endpoint values
during the later time period. For this route, figures for adjusting cardiac output
and blood flow based on brain AChE are similar to, but show more variability
than those for when adjustments are not made.
8.3.2.2 Sensitivity Analyses for the Subcutaneous Route

There are fewer parameters to which the model is sensitive for the subcu-
taneous route than for the intravenous route (Table 8.6), perhaps reflecting
the less rapid changes in endpoints due to the slower uptake of the dose.
Sensitivity time courses for blood VX concentration are similar between the
subcutaneous and intravenous routes (Figure 8.16). For AChE activity end-
points, time courses for sensitivity coefficients are fairly similar with a rise
in sensitivity followed by a decrease (Figures 8.17–8.19). The differences are
in the time period over which the rise and fall occur, with an increase in the
length of the period possibly reflecting the longer time for VX to reach the
target for each endpoint. While the sensitivities for AChE activity in the dia-
phragm have not started to decrease much by the end of the time simulation,
it is expected that they would continue in a manner consistent with the other
two AChE endpoints if the simulation were extended. Figures for not adjust-
ing cardiac output and blood flow based on brain AChE compared with those
for adjusting are almost identical.
230 Chapter 8

of (a) AChE and (b) BuChE activity, as a fraction of baseline
measurements, in whole blood of male Duncan Hartley guinea pigs fol-
lowing a single subcutaneous dose (line) of 0.8 µg VX kg−1 BW (0.1 LD50).
Triangles represent measured means ±2 standard errors of the mean.9
Inset shows simulation and data for early time points.
8.3.2.3 Sensitivity Analyses for the Dermal Route

Unlike other routes for brain and diaphragm AChE activity, with the dermal
route, the most extreme coefficient values are all smaller when adjusting for
brain AChE activity compared with not adjusting (Table 8.7). Time courses of
sensitivities for all four endpoints (Figures 8.20–8.23) are similar to those for
the other routes with the exception that the changes seem to be drawn out
over a longer time period, thus reflecting the slower uptake of VX from the
skin. Overall, patterns of sensitivities for the AChE endpoints are very similar
to those for the subcutaneous routes. As with the other routes, figures for not
adjusting cardiac output and blood flow based on brain AChE compared with
those for adjusting are almost identical.

of AChE activity in (a) blood and (b) brain of male Hartley
guinea pigs following a single subcutaneous dose (line) of 8 µg VX kg−1
BW (1 LD50). Symbols represent measured means ±2 SEMs.29–31 Dif-
ferent symbols in (a) represent data from the three different sources
(2011 = circle, 2010 = square, 2005 = triangle). Different symbols in
(b) represent measured values for different regions of the brain (in
decreasing order of activity level: striatum, spinal cord, brainstem and
cerebellum, midbrain and hippocampus, cortex).
8.4 Discussion
8.4.1 PBPK Model Structure
This PBPK-PD model for VX is based on previously developed PBPK-PD mod-
els4,6,7 describing the kinetics and dynamics from DFP or GB exposure. These
previously published models served as the starting point to which modifi-
cations were made to make the model more applicable to VX. One modi-
fication that was not made to the model was the deletion of CaE activity.
Whereas binding to CaE is much less important for VX than for G agents,33
232 Chapter 8

of blood (a) VX concentration (ng ml−1) and (b) AChE activ-
ity in male hairless guinea pigs following a single dermal dose (line)
of 125 µg VX kg−1 BW (1 LD50). Triangles represent measured means ±2
standard errors of the mean.10
both inhibition and reactivation rates have been measured in the rat;24,34
rates for guinea pigs were not located in the published literature. Even very
small amounts of CaE inhibition may have an impact on the amount of VX
available for other routes of inhibition, binding and elimination.
The first modification was the inclusion of diffusion limitation to the tissue
compartments. It is thought that larger charged molecules diffuse less readily
into tissues, thus negating the assumption of instantaneous diffusion in a flow
limited compartment.35 While VX may not be considered a large molecule, it is
larger than DFP and GB, and is mostly ionized at physiological pH. Addition of
diffusion to the tissue compartments also helps to simulate the longer clear-
ance time of VX from the blood compared with GB. The necessity of diffusion
parameters with small values (less than 1 l h−1 kg−0.75 for all but the brain) also
supports the validity of assuming diffusion in the tissues.
Table 8.5 Maximum
sensitivity coefficients for a single intravenous dose of 28 µg VX kg−1 BW.a,b
Modeling Organophosphorus Chemical Warfare Nerve Agents

Endpoint
AChE Activity in Sensitive
for another
Parameter Blood VX concentration Blood Brain Diaphragm dosing route
Body weight −0.6 (−0.6) 0.3 (−0.3) 1.2 (1.2) 0.7 (0.5) Yes
Cardiac output −2.0 (2.0) −1.4 (−1.4) −8.8 (−4.9) −2.0 (−0.3) Yes

Fractional blood flows
Brain −2.2 (—) 0.8 (—) −8.0 (−4.3) −3.7 (—) Yes
Diaphragmc 2.5 (—) No
Fatc −0.3 (—) −0.3 (—) −0.2 (—) No
Rapidly perfused 2.9 (1.4) −2.0 (−1.0) −7.3 (−0.5) −2.2 (—) Yes
Skin −0.5 (−0.2) −0.2 (−0.2) −0.5 (—) −0.4 (—) Yes
Slowly perfused 1.1 (0.5) −0.7 (0.4) −1.6 (—) Yes

Fractional tissue volumes (includes blood)
Arterial blood 1.6 (−1.0) 2.6 (2.6) 3.0 (2.0) 2.4 (0.7) Yes
Brain 0.4 (—) −0.2 (—) 5.8 (6.0) 4.9 (—) Yes
Diaphragm −1.0 (4.3) Yes
Lung −1.0 (−1.0) 0.3 (0.3) Yes
Rapidly perfused 0.7 (0.7) 0.8 (0.2) 0.5 (—) No
Slowly perfused 0.7 (—) −0.4 (—) 0.3 (0.3) 0.3 (—) Yes
Venous blood 1.4 (−1.0) 4.9 (4.8) 6.6 (4.1) 5.4 (1.7) Yes

Fractional tissue blood volumes
Diaphragmc −5.1 (—) No
Liverc 0.4 (—) 0.3 (—) No
Lung −1.0 (−1.0) 0.3 (0.3) Yes
Rapidly perfused 0.3 (0.3) 0.3 (—) No
Skin 0.2 (—) Yes
Slowly perfused 0.5 (—) −0.3 (—) — (0.2) Yes
233
(continued)
234
Table 8.5 (continued)
Endpoint
AChE Activity in Sensitive
for another
Initial concentration of B-esterase binding sites
Brain AChE 1.3 (—) −0.4 (—) 5.0 (1.6) 1.5 (—) Yes
Diaphragm AChE 1.1 (1.5) Yes
Blood BuChE − (0.2) 0.3 (—) 0.2 (—) Yes
Brain BuChEc 0.5 (—) No
Diaphragm BuChE 0.7 (1.0) No

Tissue/blood partition coefficients
Brain −1.1 (—) 0.4 (—) −3.8 (4.3) 3.4 (—) No
Diaphragm 2.9 (1.8) No

Diffusion limitation constants
Brain −0.5 (—) −1.9 (−1.4) −0.9 (—) Yes
Diaphragm −2.1 (−4.0) Yes

Maximum metabolic rate
Blood −1.9 (−1.6) 1.1 (1.1) 2.2 (1.6) 2.1 (2.3) Yes
Brain 2.4 (—) −0.8 (—) 9.3 (0.5) 0.2 (—) Yes

Chapter 8
Michaelis–Menten affinity constants
Blood 1.9 (1.7) −1.1 (−1.1) −2.0 (−1.5) −2.0 (−2.2) Yes
Brain −0.2 (−0.3) Yes
Non-B-esterase binding rates

Fast on rate 2.1 (1.5) 1.8 (1.8) 5.2 (3.8) 4.9 (1.1) Yes
Fast off rate −2.1 (−2.0) 1.1 (1.2) −0.3 (1.3) −0.4 (−1.0) Yes
Slow on rate −0.6 (−0.8) 0.4 (0.6) 1.2 (0.9) 1.1 (1.2) Yes
Slow off rate 0.7 (0.7) −0.6 (−0.6) — (−0.2) Yes

Pharmacodynamic rates
AChE inhibition 0.6 (—) −7.6 (−7.6) −3.8 (−5.7) −6.9 (−2.7) Yes
BuChE inhibition 0.6 (0.8) Yes
AChE reactivation 0.5 (—) 0.8 (0.8) 0.9 (0.8) 0.9 (0.8) Yes
BuChE reactivation −0.3 (−0.4) Yes
AChE degradation 0.2 (—) 0.2 (0.2) Yes

Dosing parameters
Length of injection −1.0 (−1.0) 1.8 (1.8) Yes
a
Numbers in the table represent the most extreme value of the time course of sensitivity coefficients for when cardiac output and blood flows were adjusted
based on AChE brain activity. Numbers in parentheses are the corresponding values for when adjustments were not made.
b
Values marked with “—” are not equal to or greater than 0.2 in absolute value, values in bold are in the high category (>0.5 in absolute value) with remain-
ing values in the medium category (≥0.2 and ≤0.5 in absolute value), and values of 0.5 that are not bolded are actually <0.5 but rounded to 0.5 for purposes
of the table.
c
The endpoints are only sensitive to these parameters for this route and only if cardiac output and tissue blood flows are adjusted based on AChE activity in
the brain.
235
Table 8.6 Maximum
sensitivity coefficients for a single subcutaneous dose of 3.2 µg VX kg−1 BW.a,b
236
Endpoint
AChE activity in
Sensitive for another
Body weight −1.0 (−1.0) −0.4 (−0.4) Yes
Cardiac output 3.0 (3.0) −0.3 (−0.3) −0.5 (−0.8) Yes

Brain −0.4 (−0.7) Yes
Rapidly perfused 3.6 (1.4) −0.3 (—) −0.4 (—) Yes
Slowly perfused 2.3 (1.5) −0.3 (—) Yes

Arterial blood −1.0 (−1.0) 0.5 (0.6) — (0.2) Yes
Brain 0.4 (0.6) Yes
Diaphragm 0.3 (0.3) Yes
Lung −1.0 (−1.0) Yes
Slowly perfused −1.0 (−1.0) 0.2 (—) Yes
Venous blood −1.0 (−1.0) 1.2 (1.2) 0.3 (0.4) Yes

Lung −1.0 (−1.0) Yes
Slowly perfused −1.0 (−1.0) Yes

Brain AChE 0.4 (0.6) Yes
Blood BuChE 0.4 (0.4) 0.2 (0.2) Yes
Chapter 8

Subcutaneous 42.6 (42.6) 0.7 (0.8) No

Brain — (−0.2) Yes

Subcutaneous 1.0 (1.0) −0.7 (−0.7) No

Blood −1.5 (−1.4) 1.5 (1.5) 0.4 (0.6) 0.3 (0.3) Yes
Brain 0.2 (0.4) Yes

Blood 1.5 (1.4) −1.5 (−1.4) −0.4 (−0.6) −0.3 (−0.3) Yes

Fast on rate 1.1 (1.2) 1.0 (1.0) 0.2 (0.3) Yes
Fast off rate −1.6 (−1.6) −0.7 (−0.7) — (−0.2) Yes
Slow on rate −0.6 (0.5) 0.6 (0.6) — (0.3) Yes
Slow off rate 0.5 (0.5) Yes

AChE inhibition −30.5 (−30.5) −2.9 (−2.9) −0.2 (−0.4) Yes
BuChE inhibition 0.3 (0.3) Yes
AChE reactivation 0.7 (0.7) — (0.2) Yes
BuChE reactivation 0.4 (0.4) Yes
AChE aging −0.2 (−0.2) Yes
AChE degradation 0.2 (0.2) Yes

Dosing parameters
Volume of dose −1.0 (−1.0) 0.7 (0.7) No
Multiple of dose −1.0 (−1.0) 0.7 (0.7) No
volume
Length of injection 42.6 (42.6) Yes
a
b
Values marked with “—” are not equal to or greater than 0.2 in absolute value, values in bold are in the high category (>0.5 in absolute value) with remain-
ing values in the medium category (≥0.2 and ≤0.5 in absolute value), and values of 0.5 that are not bolded are actually <0.5 but rounded to 0.5 for purposes
237
of the table.
Table 8.7 Maximum
sensitivity coefficients for a single dermal dose of 60 µg VX kg−1 BW.a,b
238
Endpoint
AChE activity in
Blood VX Sensitive for another
Parameter concentration Blood Brain Diaphragm dosing route
Body weight −1.4 (1.7) 1.4 (1.7) 0.3 (0.7) 0.3 (0.6) Yes
Cardiac output 3.0 (3.0) −1.5 (−2.0) −0.6 (−1.6) −0.3 (−0.8) Yes
Skin thickness −0.8 (−0.8) −0.4 (−0.5) — (−0.3) — (−0.3) No

Brain −0.2 (—) 0.3 (—) −0.2 (−0.6) Yes
Rapidly perfused 3.6 (1.4) −1.1 (—) −0.5 (—) −0.3 (—) Yes
Skin 1.0 (1.0) −1.6 (−2.0) −0.3 (−1.0) −0.4 (−0.8) Yes
Slowly perfused 1.3 (0.5) −0.4 (—) Yes

Arterial blood −1.0 (−1.0) 0.2 (0.3) Yes
Brain — (0.3) Yes
Diaphragm 0.5 (0.8) Yes
Lung −0.9 (−0.9) Yes
Skin −1.0 (−1.0) 1.6 (2.0) 0.3 (1.0) 0.4 (0.8) No
Venous blood −1.0 (−1.0) 0.5 (0.6) — (0.2) Yes

Lung −0.9 (−0.9) Yes
Skin −0.8 (−0.8) Yes

Brain AChE — (0.3) Yes
Chapter 8
Blood BuChE 0.2 (0.2) Yes

Dermal −1.0 (−1.0) 1.7 (2.1) 0.3 (1.0) 0.4 (0.8) No
Skin/saline partition 0.3 (0.4) −0.4 (−0.5) — (−0.3) — (−0.3) No

Brain — (−0.2) Yes
Dermal 0.8 (0.8) No

Blood −1.2 (−1.3) 1.0 (1.2) 0.2 (0.7) 0.4 (0.6) Yes
Brain 0.2 (0.6) Yes
Skin −7.0 (−7.9) 1.9 (2.5) 0.5 (1.3) 0.6 (1.1) No

Blood 1.2 (1.3) −0.9 (−1.1) −0.2 (−0.7) −0.3 (−0.6) Yes
Brain −0.2 (−0.6) Yes

Fast on rate −0.4 (−0.4) Yes
Slow on rate 0.6 (0.5) 0.4 (0.5) — (0.3) — (0.2) Yes
Slow off rate 0.5 (0.5) — (−0.2) Yes

AChE inhibition −0.2 (—) −1.7 (−2.0) −0.2 (−0.7) Yes
AChE reactivation 0.7 (0.8) — (0.3) Yes
AChE aging −0.3 (−0.3) Yes
AChE degradation 0.3 (0.3) Yes

Dosing parameters
Permeability 1.1 (1.1) −1.0 (−1.3) — (−0.4) — (−0.3) No
Exposure area 1.1 (1.1) −1.3 (−1.7) −0.2 (−0.7) −0.3 (−0.6) No
Initial concentration 1.1 (1.1) −1.4 (−1.7) −0.2 (−0.7) −0.3 (−0.6) No
on skin
a
239
b
Values marked with “—” are not equal to or greater than 0.2 in absolute value, values in bold are in the high category (>0.5 in absolute value) with remaining val-
ues in the medium category (≥0.2 and ≤0.5 in absolute value), and values of 0.5 that are not bolded are actually <0.5 but rounded to 0.5 for purposes of the table.
240 Chapter 8
Figure 8.12 Selected

time courses of sensitivity coefficients for the endpoint of
blood VX concentration following a single intravenous dose of 28 µg
VX kg−1 BW with cardiac output and tissue blood flows adjusted for
brain AChE activity.
Another modification to the model was the addition of non-B-esterase

binding in the blood. Limited information was found on in vivo serum albu-
min binding of VX in rats and guinea pigs36 and in vitro binding to human
blood.37 There is also additional evidence of organophosphates and other
nerve agents binding to human serum albumin (e.g., DFP, chlorpyrifos oxon,
GB and GD)38–40 and guinea pig albumin (GB, GD, cyclosarin and GA).37 Duy-
sen et al.41 published a study on the lethality of VX in AChE knockout mice
that indicates toxicity in wild-type mice is likely due to binding to several pro-
teins other than AChE. Simulations of the intravenous data of van der Schans

blood AChE activity following a single intravenous dose of 28 µg VX
kg−1 BW with cardiac output and tissue blood flows adjusted for brain
AChE activity.
et al.10 also indicate the need for simulations of additional binding of VX in

order to duplicate the multi-phasic nature of the data. While increasing the
bimolecular inhibition rates for AChE and BuChE decreases the predicted
blood concentrations, it also increases the predicted inhibition of AChE
and BuChE, thus over-predicting inhibition. It was also necessary to incor-
porate non-B-esterase binding through two sets of terms to represent both
rapid and slow binding, as a single set of parameters is unable to adequately
simulate the data. While these parameters are not likely to be measured for
any species, they can be estimated by fitting the model to the available data;
242 Chapter 8

brain AChE activity following a single intravenous dose of 28 µg VX
AChE activity.

diaphragm AChE activity following a single intravenous dose of 28 µg
some data are available in the published literature for other species, includ-
ing for humans.
There are numerous PBPK models that simulate dosing via subcutaneous
or dermal routes, but not all create a dosing compartment specific for the
route and separate from other tissue compartments. While not using sep-
arate compartments is a simpler method of simulating these dose routes,
it also necessarily assumes that the dose is in equilibrium within the com-
partment to which it is introduced (i.e., the entire skin compartment rather
than a small piece of the skin compartment). Intravenous dosing is typically
handled in this manner as well; however, since there is no uptake compo-
nent to this dose route, it probably has little impact on model predictions
versus the data. For other routes, uptake from the dosing site is dependent
on compartment concentration and, thus, the compartment volume. By
244 Chapter 8

blood VX concentration following a single subcutaneous dose of 3.2
µg VX kg−1 BW with cardiac output and tissue blood flows adjusted for
brain AChE activity. Inset in (d) is the same data just on a shorter time
scale to see the smaller values of the coefficients. Note that some lines
completely overlap: (a) lines for fractional lung volume and blood in
lung; (d) lines for length of injection and subcutaneous/blood parti-
tion coefficient until the end of the injection; (e) lines for dose volume
and multiple of dose volume. Coeff: coefficient.
using a non-dose-specific compartment to receive the dose (e.g., the entire

slowly perfused compartment for a subcutaneous dose), the concentration
for uptake is underestimated by the use of a larger compartment volume.
By creating dose route specific compartments, a more appropriate volume
may be used so that uptake may be more adequately simulated. The PBPK-PD

blood AChE activity following a single subcutaneous dose of 3.2 µg VX
AChE activity.
246 Chapter 8

brain AChE activity following a single subcutaneous dose of 3.2 µg VX
AChE activity.
Figure 8.19 Time

courses of sensitivity coefficients for the endpoint of diaphragm
AChE activity following a single subcutaneous dose of 3.2 µg VX kg−1
BW with cardiac output and tissue blood flows adjusted for brain
AChE activity.
model presented here goes a step further and only creates these dose route
specific compartments as needed. These dosing compartments are created
from existing tissue compartments by subtracting a volume representative
of the dosing site from the existing tissue compartment. It is also worth not-
ing that this method is also a simplification as it does not account for the
fact that the volume of the dosing compartment may change over time as
the dose is introduced and subsequently absorbed. This method is, however,
more biologically accurate than the method of not using a separate dosing
compartment, and has been successfully demonstrated in a model published
by Sweeney et al.19

blood VX concentration following a single dermal exposure of 60 µg
The model description of dermal dosing, while common in PBPK models,

is an over-simplification of the process. It assumes that VX moves directly
through the skin surface into the underlying tissue, whereas the actual path-
way is probably much more complex, such as a transcellular route through
the hydrophilic corneocytes and the lipophilic matrix between the corneo-
cytes, or an intercellular route passing only through the matrix between the
corneocytes.42 Lateral diffusion in the skin is also a possibility and several
studies have demonstrated that VX is stored just under the skin surface,
not yet absorbed but no longer on the surface.13 The model also does not
248 Chapter 8

blood AChE activity following a single dermal exposure of 60 µg VX
AChE activity.

time courses of sensitivity coefficients for the endpoints of
brain AChE activity following a single dermal exposure of 60 µg VX
AChE activity.

time courses of sensitivity coefficients for the endpoints of
diaphragm AChE activity following a single dermal exposure of 60 µg
currently allow for a lag in time before steady-state permeability is reached.

Some studies have reported the lag time before this steady-state is reached;43
however, how the permeability changes during this lag period is not reported,
thus making it difficult to incorporate it into a model.
Another issue in describing dermal dosing is the dependence of the
absorbed dose on skin thickness and blood flow to the skin in the exposed
area. While the model does allow for the skin thickness to be altered for a
given simulation and blood flow to the exposed area may be adjusted, data
for these parameters are quite often only available for limited anatomical
250 Chapter 8
locations. The absorbed dose can also be affected by the spread of the applied
liquid and the vehicles used to dilute VX. The average area of spread per mg
of VX may vary across and even within species,13 but is typically not reported.
With guinea pigs, it is necessary to dilute VX in order to dose in small enough
quantities to avoid death of the animal; however, these solvents can affect
the absorption of the applied chemical,43,44 thus skewing the experimental
results. Variations in all of these aspects could result in variations in sim-
ulated endpoints from non-lethal inhibition to lethal inhibition of AChE,
where the actual data lie somewhere in between.
The model is coded with the capability to adjust cardiac output and tissue
blood flows based on the level of brain AChE activity in order to mimic the
physiological response to AChE inhibition. While this is not incorporated
into the previously published models for DFP and GB, it is included here
in an effort to incorporate available data on decreasing minute ventilation
following dermal exposure to VX.45 This study reports a decrease in minute
ventilation that begins approximately 30 min after exposure, which is approx-
imately the same time that cholinesterase (ChE) activity in the blood drops
to below about 20% of the baseline value. Given the correlation between ven-
tilation and cardiac output, it is assumed that cardiac output, and thus tis-
sue blood flows, would also decrease. This assumption is supported by data
indicating decreases in various tissue blood flows in rats following intrave-
nous exposure to paraoxon,46 and decreases in cardiac output in cats and rab-
bits following exposure to paraoxon.47,48 While Maxwell et al.8 did not note
a decrease in cardiac output in rats following paraoxon exposure, they did
note decreased blood flow to some tissues, indicating peripheral vasocon-
striction, and stated that this vasoconstriction is common in various species
but is unique in rats in that it is not also accompanied by decreased cardiac
output. It is also assumed that the effect of decreased minute ventilation
was more closely related to brain AChE activity than blood activity. Since the
study only reported blood activity levels and decreases in ventilation, a quan-
titative relationship between cardiac output and brain AChE activity could
not be incorporated into the model. In an effort to approximate this rela-
tionship, the model is coded to allow cardiac output to decrease by the same
fraction that brain AChE decreases over time. This adjustment is also coded
such that it could be turned on or off depending on the exposure scenario
being simulated.
Prior to the inclusion of metabolism in the skin compartment, the model
was unable to accurately simulate the dermal dosing data of Lumley et al.9
(60 µg VX kg−1 BW); accurate prediction of the amount of VX remaining on
the skin resulted in over-prediction of blood VX concentration and blood
AChE activity. If model parameters were adjusted to adequately predict blood
concentration and activity, the amount on the skin was no longer well pre-
dicted. While no specific information was found for skin metabolism of VX,
Carver et al.49 noted metabolism of another organophosphate, parathion, in
isolated perfused porcine skin flaps. Inclusion of skin metabolism is also
supported by the much improved model predictions of both the dermal
datasets without the necessity to adjust any model parameters other than
experiment specific ones. Also, the sensitivity of both blood VX concentra-
tion and blood AChE activity to the maximum skin metabolism rate, along
with the improved model predictions, tends to support the importance of
the addition of this metabolic rate for the prediction of these endpoints. It
should also be noted that this metabolism parameter is only sensitive for
the dermal route, indicating that it has little impact on the other two dosing
routes.
8.4.2 Model Parameters

While blood flows are often readily available from the literature, they are
not available for dosing compartments as they are arbitrary and specific
to the model in which they are used. However, models scaling blood flow
for a compartment based on a fractional relationship involving its volume
have been published.50–52 Blood flows to the separate dosing compartments
are calculated as a portion of the flow to the parent compartment (skin or
slowly perfused) based on the ratio of the volumes of the dosing and parent
compartments.
Length of injection is not typically reported in studies unless the injec-
tion is given as an infusion. It is typically set to a small time value and is
often fit to the data. For the simulations presented here, length of injection
for both the intravenous and subcutaneous routes is set to a small value. To
support the choice of this value, this parameter was included in the sensi-
tivity analyses. Model predictions for blood VX concentration for the intra-
venous and subcutaneous routes are sensitive to length of injection, but
only during the time of injection, and are most sensitive at the beginning
of the injection with a spike later at the end of the injection. Predictions for
AChE activity in blood for the intravenous route are also sensitive to length
of injection; however, the sensitivity in this case is zero until just prior to the
end of the injection period and peaks, in absolute value, at a later time than
for blood concentration with the largest sensitivity occurring a few seconds
after the end of dosing. It is not unexpected that sensitivities of AChE activ-
ity would peak later than those for blood concentration as activity levels are
not affected as quickly as blood levels. It is important to note, however, that
sensitivity coefficients in all three cases decrease to less than 1 in absolute
value before 1 min.
There is no preferred value to use in defining the volume of the subcu-
taneous dosing compartment. Sweeney et al.19 defined their subcutaneous
dosing compartment with a deposit compartment for the dose itself that was
set equal to the volume of the injection and a subcutaneous tissue compart-
ment with a volume set equal to 10 times the volume of the injection. The
PBPK-PD VX model has a simpler description with a single compartment and
a volume set at twice the injected volume. As with length of injection, this
parameter (representing the factor of 2) was included in the sensitivity anal-
yses. Only blood VX concentration and blood AChE activity were sensitive
252 Chapter 8
to this parameter; however, sensitivities were all less than or equal to 1 in
absolute value and decreased to almost zero by 1 h. This indicates that the
relationship between endpoint and parameter is no more than a 1 to 1 rela-
tionship. Since increasing the volume decreases the concentration in the
subcutaneous compartment and effectively acts like a dose decrease, this is
a behavior that would be expected. This concept is reinforced by the fact that
sensitivities for this parameter are the same as those for the volume of the
dose. Therefore, although the choice of setting the volume to twice the injec-
tion volume may seem arbitrary, it is much less important for predicting the
uptake of the dose than the blood flow or tissue/blood partition.
Tissue/blood partition coefficients for the dosing compartments are
defined independently of the compartments from which they are subtracted
to allow for dilutions of the doses that might result in differences in uptake
kinetics. The model is unable to predict the data for the three different sub-
cutaneous doses using a single value for the subcutaneous dosing compart-
ment. Since all of the subcutaneous doses were diluted such that the same
amount of solution was injected for all three doses, it is assumed that a
smaller dose would have a smaller partition given that a smaller dose would
mean a larger proportion of saline injected into the dosing compartment,
which would then increase the fraction of water in the tissue, thus decreasing
the predicted partition for VX. Given that the subcutaneous dosing compart-
ment does not correspond to a real tissue, the partition for the smaller dose
is estimated based on fitted values for the two larger doses rather than trying
to predict a value using QSAR. In the case of the dermal dosing compart-
ment, since the dose is absorbed rather than injected, it is assumed that the
dosing compartment tissue composition would not vary based on dilution of
the dose but that partitioning from the skin into the blood at the dosing site
would vary from partitioning in the rest of the skin due to the presence of
isopropanol. Even though the dilution of the two dermal doses was different,
it is not necessary to use different partition values for the them.
Several of the pharmacodynamic parameters were fit to the data either
because no data were located for the parameter or because use of the value
from the literature in the model did not provide adequate fits. When pub-
lished values were available, they were used as starting values for the param-
eters that were ultimately fit in order to better simulate the data. While values
for bimolecular inhibition of AChE by VX are available in the literature for
guinea pigs,10,53 these values were measured in vitro and neither adequately
simulates the in vivo inhibition data. With in vitro measured values, there
is uncertainty as to how accurately they represent in vivo processes as the
experiments are not always conducted in the presence of acetylcholine or
other esterases and proteins to which VX might bind, thus reducing the rate
at which it would bind to AChE. It is also unclear in some studies whether it
is the actual unidirectional bimolecular reaction rate that is being measured
or if it is a composite rate that includes both the unidirectional bimolecular
rate and the reactivation rate. The fit value used in the model for AChE is
about 70% of the value measured by van der Schans et al.,10 but is also only
slightly larger than an in vitro value measured for humans by Ashani and
54
Pistinner. While the model was sensitive to this parameter for all routes
(see further discussion below), these sensitivities are not illogical and the
value used did quite well at predicting all of the data with the exception of
data for a subcutaneous dose of 0.8 µg VX kg−1 BW.
While the model is sensitive to BuChE bimolecular inhibition and reacti-
vation rates, the only other fit pharmacodynamic rates, sensitivities for all
but diaphragm AChE activity following an intravenous dose were medium
or lower. Measured data on these two parameters in guinea pigs could not
be located. While the fit bimolecular inhibition rate used in the model for
BuChE is about half of a measured rate in humans,54 it should result in more
conservative estimates for AChE inhibition by allowing for more free VX due
to less binding to BuChE. A measured reactivation rate for BuChE in humans
was also located;55 this value is smaller than the fit value used in the model,
but should also result in more conservative estimates for AChE inhibition.
The reactivation and degradation rates used in the model for CaE are the
same as those used for AChE, and the model is not sensitive to these parame-
ters for any endpoint for any dose route. The CaE reactivation value is about a
factor of 10 smaller than the measured value for rats found in the literature,24
but a measured value for guinea pigs could not be located. A degradation rate
for CaE in guinea pigs could not be found either. There were no data with
which to directly validate parameters for CaE.
8.4.3 Simulations
The model presented here does quite well at simulating the available data.
While some of the data are under- or over-predicted, these deficiencies might
be corrected by changing some of the model parameters to more accurately
describe the particular strain of guinea pig being used in the individual stud-
ies. It is also important to note that some of the animals in these studies
received atropine prior to exposure, which probably altered the pharmaco-
kinetics and pharmacodynamics of VX; however, given the small amount of
data available, it was felt that exclusion of these data would be more detri-
mental than inclusion. The model simulations, in comparison to available
guinea pig data, verify that VX is poorly metabolized, such that, unlike GB or
GD, it lingers in the blood long enough and at high enough concentrations
to be quantified. This complex multi-component clearance process from
the blood could indicate the possible presence of VX reservoirs in the body.
Whereas the model incorporates non-B-esterase binding to account for part
of this clearance process, it could be an over-simplification of what is actually
occurring.
This model has several strengths, particularly its ability to simulate the
recovery of AChE and BuChE as well as tissue AChE activity, especially brain
and diaphragm AChE activity. AChE inhibition in the central nervous system
is widely considered a primary target of nerve agents such as VX and, there-
fore, it is vital for a PBPK-PD model for nerve agents to accurately simulate
this process. Since VX differs from G agents in several areas, it is also possi-
ble that it may act directly on peripheral targets, such as the diaphragm, in
254 Chapter 8
addition to the central nervous system. Thus, it is also important to point
out that the model does quite well at predicting AChE activity in this tissue as
well. Another important strength is the model’s ability to quite successfully
predict both dermal datasets without any changes to any parameters other
than those that are experiment specific (BW, dose, exposure area and initial
concentration on the skin) even though there are differences in strain, dos-
ing site and dilution rate between the two different experiments.
Although the model presented here does a good job of simulating the available
data, it also helps highlight areas where further improvements may be made.
The data for subcutaneous dosing at 0.8 and 3.2 µg VX kg−1 BW point out that an
increase in the dose by a factor of 4 did not result in a similar decrease in AChE
activity. The model accounts for part of this difference, but it still under-predicts
activity at the lower dose. While the value used for the subcutaneous dosing
compartment partition is predicted for the lower dose, a larger partition than
that used for the higher dose is needed to correct the under-prediction; however,
with the higher partition, the model then over-predicts earlier time points and
cannot predict as rapid a drop in activity as is indicated by the data. This failure
to predict the lower subcutaneous dose could be simply an issue with the actual
baseline AChE levels for these guinea pigs differing from the levels used in the
model or it could indicate a potentially saturable process for which the model
is either not accounting for or is not accurately parameterized. These processes
could be non-B-esterase binding, metabolism or urinary excretion. There are no
data with which the currently coded non-B-esterase binding could be directly
validated and binding rates were not located in the published literature. Also,
while there are limited data for EMPA, there are no other data to definitively
determine if, for the guinea pig, EMPA is the only metabolite.56 Thus, metabo-
lism parameters were indirectly fit to blood concentration data. There are also
limited data on VX concentration in the urine; however, the reported data are
for concentration in the urine at given time points without any corresponding
volumes of urine. Urinary excretion rates are more accurately fit to data for the
amount of cumulative excretion.
Another possible improvement in the model could be in the simulation of
CaE activity. As mentioned above, VX binding to CaE is much less important
than for G agents;33 however, not including it in the model could have an
impact on the amount of VX available for other routes of inhibition, bind-
ing and elimination. Code is included in the model to account for CaE activ-
ity, but parameter values for the guinea pig for this enzyme were not readily
available from the literature and data are not available for direct validation
of this portion of the model.

Clewell et al.32 state that sensitivity coefficients larger than 1 in absolute
value could be worrisome as they represent “amplified error”. They also
note that substantial fluctuations in sensitivity calculations could be a result
of numerical imprecision when simulated endpoint values are very small.
While there are numerous instances in these analyses of coefficients larger
than 1 in absolute value, these values occurred, for all routes, at points of
either rapidly changing endpoint values or very small endpoint values. This
behavior is more easily seen when the endpoint and sensitivity coefficients
are plotted on the same figure (Figures 8.24–8.26). These increased sensitivi-
ties could, therefore, partly be a result of numerical imprecision, as opposed
to just model sensitivity.
For injected routes (intravenous and subcutaneous), blood VX concentra-
tion was generally most sensitive during the injection period when blood lev-
els are rapidly changing or towards the end of the simulation when blood
levels are very small. The pattern for the dermal route is similar with the larg-
est sensitivities occurring when blood levels are very low and during the early
uptake of the dose until about 2 h; however, the metabolism parameters are
the exception with sensitivities to these parameters peaking towards the end
of the simulation when the amount in the dermal dosing compartment is
very small. This exception is also true for the skin maximum metabolic rate
for the endpoint of blood AChE activity for the dermal route, but sensitivities

of time courses for endpoints [(a) blood VX concentra-
tion, (b) blood AChE activity, (c) brain AChE activity and (d) diaphragm
AChE activity] and time courses for sensitivity coefficients for selected
parameters following a single intravenous dose of 28 µg VX kg−1 BW
with cardiac output and tissue blood flows adjusted for brain AChE
activity.
256 Chapter 8

parameters following a single subcutaneous dose of 3.2 µg VX kg−1 BW
activity.
to all other parameters for this endpoint for all routes are generally largest as
endpoint values drop to near their lowest values and while the endpoint is at
its lowest values (i.e., near zero). Brain and diaphragm AChE activities for the
intravenous route showed sensitivities similar to those seen for blood AChE
activity, but were all between 1 and −1 for the subcutaneous and dermal
routes. The instances of extremely large sensitivity coefficients (i.e., larger
than 10 in absolute value) were all for the subcutaneous route and occurred
early in the simulation when blood levels were practically zero.
In general, the selected model endpoints are sensitive to the largest num-
ber of parameters for the intravenous route. This is not surprising because
the blood concentration changes more rapidly with this dose route due to
the much more rapid uptake of the dose. It is also not surprising that the
endpoints of AChE activity in the brain and diaphragm are sensitive to more
parameters than the endpoints of AChE activity in the blood and blood VX
concentration for the intravenous route since the brain and diaphragm end-
points are dependent on various parameters governing transport to these
tissues. It is, however, somewhat surprising that brain and diaphragm AChE
activity are sensitive to fewer parameters for the subcutaneous and dermal

parameters following a single dermal exposure of 60 µg VX kg−1 BW
activity.
routes; this could perhaps be due to the slower uptake and, hence, less rapid
changes in blood concentration values for these two routes.
For the subcutaneous route, only two of the endpoints (blood VX concen-
tration and blood AChE activity) are sensitive to dose volume and the mul-
tiple of dose volume; however, sensitivities to these two parameters are less
than 1 in absolute value for both endpoints. Given that a change in these two
parameters effectively acts as a change in dose, one would expect something
of a 1 to 1 relationship between these parameters and endpoints, at least
during the period of uptake. A similar behavior is seen for these two end-
points for the dermal route for the parameters of exposure area and initial
concentration on the skin; however, the sensitivities to these two parameters
does peak slightly above 1 in absolute value.
For injections that are not infusions, the length of the injection is not usu-
ally reported in studies; therefore, it is normally set to a small time value and/
or fit to the data. For the simulations presented here, length of injection for
both the intravenous and subcutaneous routes is set to a small value. To sup-
port the choice of this value, this parameter was included in the sensitivity
analyses.
258 Chapter 8
As the length of injection was not stated for the studies, a small value was
used for both the intravenous and subcutaneous simulations. While large
sensitivities to this parameter are seen, they occur during time periods when
the blood concentration begins to change rapidly; thus, larger calculated
sensitivity coefficients could be due to some numerical instability and could
potentially be decreased by using an even smaller communication interval
for this simulation period. These sensitivities could also be artifacts of how
intravenous and subcutaneous dosing are described in the model.
It is interesting to note behaviors exhibited by the time courses for the
sensitivity coefficients such as the parameters that have similar shapes, dif-
fering significantly only in magnitude, and those whose curves are mirror
images of each other. These patterns could possibly indicate relationships
between these parameters that might not initially be obvious, such as sensi-
tivity to a parameter being not so much a direct result of that parameter on
the predicted endpoint but rather the impact on the endpoint of a different
parameter that is influenced by changing another parameter. The sensitivity
analyses also show that changes to several parameters can result in the same
impact on the specified endpoint, indicating that the model may be sensitive
to a given parameter in as much as it is sensitive to changes in a given process
(e.g., transport).
8.5 Conclusions
This manuscript describes the development of a PBPK-PD model for VX expo-
sure and parameterization for guinea pigs. The model, in general, does a good
job of simulating both the data used for development and the validation data
from three routes of exposure. The intravenous route was included in the
model as this route allows non-dose-route specific parameters to be more
easily estimated without accounting for complications due to the dose route.
The subcutaneous route was then added as it serves as a good surrogate for
dermal dosing without the uncertainties that exist in accurately describing
uptake via the dermal route. Lastly, the dermal route was included as it is the
most likely real world exposure route for VX. Although additional data could
allow for further refinement of the current model, the model presented here
represents a sound basis for expansion to other species, including humans.
The ability to extrapolate to VX exposure in humans would allow for improve-
ment of current risk estimates for VX exposures and can serve as a useful
means to study potential therapeutics. Until now, a PBPK-PD model for VX
has not been published.
Acknowledgements
This work received support from the Defense Threat Reduction Agency—
Joint Science and Technology Office, Basic and Supporting Sciences Division
(CBM.NEURO.03.10.AHB.005).
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Chapter 9
Allometric Modeling of
Mammalian Cyanogen Chloride
Inhalation Lethality
Douglas R. Sommerville*a
a
US Army Edgewood Chemical Biological Center, 5183 Blackhawk Road,
ATTN: RDCB-DRI-M, Aberdeen Proving Ground, MD, USA 21010-5424
*E-mail: douglas.r.sommerville.civ@mail.mil
9.1 Introduction
West et al. (1997)1 and others2–4 have noted that an organism’s size affects
the rates of all biological structures and processes from cellular metabo-
lism to population dynamics. Thus, it is not surprising that allometry—the
study of the relationship of body size to shape, anatomy and physiology—
has been extensively used in toxicology for scaling of the effective dose
between species5–10 and within species.11,12 For most exposure routes,
the measurement of the delivered dose (via direct injection or feeding)
is a straightforward matter. However, in inhalation (IH) toxicology, the
dose received is directly related to the amount of air inhaled,13–15 and
the amount of inhaled air per unit time also follows an allometric func-
tion.4 Thus, the allometric modelling of IH toxicology between species is
complicated by both the effective and delivered doses being allometric

264
Allometric Modeling of Mammalian Cyanogen Chloride Inhalation Lethality 265
functions (unlike other exposure routes). Despite this, allometric mod-
elling has previously been used in developing human acute IH lethality
estimates.8,16–22
In this chapter, the allometric modelling of a chemical’s acute IH lethal-
ity will be demonstrated using cyanogen chloride, with a procedure previ-
ously used for chlorine (with some modification).17,18 Cyanogen chloride
(with the military designation of CK)23 was first synthesized in 1802 by
the French chemist, Claude Louis Berthollet.24 It is one of several cya-
nide compounds (hydrogen cyanide, cyanogen bromide, cyanogen iodide
and CK) that have been investigated for use as chemical warfare (CW)
agents, and it was first used in 1916 by the French during World War I
(WWI) in that role,24–27 although its actual effectiveness in the field was
a subject of debate.† After WWI, CK was one of two cyanide compounds
(the other being hydrogen cyanide—military designation AC)23 that were
still seriously considered as potential CW agents up until World War II
(WWII).† 24,28 CK toxicity was extensively investigated for this role during
both WWI and WWII by the UK and the USA (see also Sections 9.3 and
9.4).† 28 The information on these investigations has long since been
declassified without fanfare, and received little notice by non-military
researchers.29
Unlike other closely related cyanide compounds, CK is not used exten-
sively in industrial manufacturing or synthesis processes, although it may
be detected as a trace pollutant in water sources as a consequence of chlori-
nation.30 However, the National Institute for Occupational Safety and Health
(NIOSH) has estimated that 1391 workers were potentially exposed in the
workplace to CK in the USA through IH and dermal contact with CK where it
was produced or used [National Occupational Exposure Survey (NOES) Sur-
vey 1981–1983].31,32
9.2 Statistical Background

MINITAB™33 (and other software) was used to estimate the parameters
of interest via several more refined statistical methods, including itera-
tively reweighted least squares (IRLS),34–36 maximum likelihood estimation
(MLE),36,37 probit analysis38 and ordinal regression.‡,§ 39–42 The exact proce-
dures are discussed below.
†
John Jenner, Principal Toxicologist, Defence Science and Technology Laboratory (DSTL)—Porton,
Porton Down, Wilshire, UK, personal communication, archival WWII records of the Chemical
Defence Experimental Establishment, Porton Down, 2014–2015.
‡
Technically, both probit analysis and ordinal regression are a type of MLE procedure. The con-
vention adopted in this paper is to use MLE to refer to calculations involving the estimation of
only one parameter (i.e. a one-factor MLE), while probit analysis will denote a multi-factor MLE
procedure.
§
Ordinal regression is also known as categorical regression.
266 Chapter 9
9.2.1 Allometric Modeling and IH Toxicology

The dependence of a biological variable X on body mass M is typically char-
acterized by an allometric scaling law of the form:

X = X0 Mb (9.1)

where b is the scaling exponent and X0 a fitted constant.1–3 These two coeffi-
cients can be estimated via the linear regression fit of X versus M on a log–log
scale, where the slope and y-intercept of the resulting line equal b and log(X0),
respectively. For biological parameters (such as blood volume and oxygen dif-
fusing capacity) that scale as a function of the volume of the animal, b is pre-
dicted and generally observed (from experimental data) to equal 1.1
In IH toxicology, the dose received by an animal is directly related to the
amount of air inhaled.13–15 Minute volume (VM) is a commonly used respira-
tory measurement, and Bide et al. (2000)4 have reviewed the existing mam-
malian VM data (as well as species body mass) for the purpose of supporting
IH toxicology research. Table 9.1 provides VM and M values from Bide et al. for
several mammalian species (non-anesthetized) that were used for allometric
modelling in this chapter. An empirical relationship (for both anesthetized
and nonanesthetized animals) of VM as a function of M has also been devel-
oped. Based on allometric scaling law, VM should scale to the 0.75 power, but
Bide et al. found that VM scales to the 0.809 power [with standard error (SE)
of 0.01 for this value] based on the available experimental data for nonanes-
thetized, adult mammals:

= VM X= VM
bv
( 0.499 ) M (0.809) (9.2)

where VM is in l min−1, M is in kg, and XV and bV are the fitted allometric
coefficients for minute volume. Bide et al. calculated a value of 15.5 l min−1
Table 9.1 Summary

of average experimental mammalian (nonanesthetized) body
mass and minute volume values used in the present work [from Bide et
al. (2000)4].a
Species Body mass (kg) VM experimental (l) VM allometric fit (l)
Mouse 0.0207 0.0310 0.022
Rat 0.261 0.203 0.168
Guinea pig 0.323 0.182 0.200
Rabbit 2.77 1.453 1.138
Cat 3.36 0.845 1.330
Monkey 4.27 1.40 1.615
Dog 13.0 4.230 3.974
Goat 36.9 9.600 9.243
a
or linear regression fits, units of m3 were used for VM instead of l. Also, experimental VM val-
F
ues were used [rather than allometric fits from Bide et al. (2000)4]. Both experimental VM and
body mass values were taken from Table 2 of Bide et al.4
for a 70 kg man from eqn (9.2). This is very close to the standard value of 15
l min−1 for light activity used by the US Department of Defense (DoD) as a
basis for their toxicity estimates for a 70 kg soldier.23 Bide et al. further noted
that their equation is for normally breathing, nonanesthetized mammals
engaged in mild activity; it is not a resting value.
For typical whole body exposures, the exact inspired amount is usually not
measured. In its place, a nominal dose (mg) can be calculated by multiplying
the lethal IH dosage, LCt50 (mg min m−3), by VM (m3 min−1):16,17,43

LD50 = VM × LCt50 (9.3)

where LD50 is the nominal dose (mg) lethal to 50% of exposed individuals
and LCt50 is the lethal concentration–time (or IH dosage) to 50% of exposed
individuals. In some cases,20,21 the LCt50 has been used in place of the LD50 as
the “dose” to be scaled (the statistical reason for which is explained below).
However, the LCt50 is worthless as a measure of the dose received without
the proper consideration of VM. All derived LCt50s have an assumed (implicit
or explicit) VM value associated with them—in the case of the US military,23
the assumed human VM equals 15 l min−1 for light activity. Changes in the
assumed VM require an inversely proportional change in the LCt50 (i.e. dou-
bling the VM requires halving the LCt50) because the underlying LD50 from
eqn (9.3) has not changed.
Toxicity is a function of many factors, and some of these (i.e. metabolic,
circulatory, etc.) may also scale allometrically in their effect. Allometric mod-
eling can be used to empirically account for the cumulative effect of these
factors and scale the nominal LD50 (mg) as a function of species body mass,
as shown in eqn (9.4). A human estimate can be obtained from mammalian
data by extrapolating to a 70 kg human.17,18
LD50 = X LD M bLD
or (9.4)
=
log ( LD50 ) log ( X LD ) + bLD log ( M )
where XLD and bLD are the fitted allometric coefficients for the LD50. In eqn
(9.4), the regressor term (LD50) and predictor term are non-independent,
with M found in both. Controversy, dating back to 1897,44 exists over regres-
sions between non-independent parameters, and it continues to this day.45–47
Such regressions often return spurious correlations. Hence, the previously
mentioned use of the LCt50 in place of the LD50 [in eqn (9.4)] was done to
avoid this issue. However, using the LCt50 as the regressor loses information.
As listed in Table 9.1, experimental species’ VM values do differ slightly from
their allometric fit. LD50s based on the allometric fit for VM will differ from
those based on experimental averages. Regressing the LCt50s will be equiva-
lent to using allometric fit based LD50s.
268 Chapter 9
Instead of replacing the LD50 with the LCt50, a better solution to the prob-
lem of non-independent parameters is to split the LD50 into two suitable
components:

log(LD50) = log(LC50) + log(tVM) (9.5)

where LC50 is the lethal concentration for 50% of exposed individuals and
(tVM), is the total volume of air inhaled during the exposure. Inserting eqn
(9.5) into eqn (9.4) and rearranging produces:

log(LC50) = k0 + kM log(M) + ktV log(tVM) (9.6)

where the k’s are fitted coefficients. The toxic load exponent (TLE; discussed
in the next section) equals the negative inverse of ktV . Both sides of eqn (9.5)
are now independent of each other, and the experimental VM information is
retained in a relevant term (total inhaled air volume). This first point rep-
resents an improvement over the LD50 approach used by Sommerville et al.
(2009, 2010) for chlorine.17,18 Two competing factors are represented in eqn
(9.6) (since kM and ktV will always have opposite signs): M and (tVM). If kM = ktV
× bV, then the dependence of the LD50 (mg) on M is due only to the species’
mass dependence of VM [eqn (9.2)], with the result being that the LC50 is inde-
pendent of M. However, if kM ≠ ktV × bV, then some M dependent process (i.e.
detoxification, etc.) other than VM is a factor.
9.2.2 IH Dose–Response Statistics and the Toxic Load

The statistical properties of the dose–response distribution have been exten-
sively reviewed48,49 and summarized.14,50 An IH dosage traditionally has been
expressed as the product of vapor concentration (C) and duration (t), or Ct.
The distribution of effective Cts for a homogeneous population is usually
log-normal and described by:

Z = m[log(LCtXX) − log(LCt50)] (9.7)

where Z is the standard normal random variable, m is the probit (or Bliss)
slope (PS; and equal to the inverse of the standard deviation), LCtXX and
LCt50 are the Cts that will produce lethality in XX% and 50%, respectively, of
the exposed individuals. Z is a function of the cumulative probability, with
the 16, 50 and 84% response level corresponding to Z = −1, 0 and 1, respec-
tively.38,51–53 Toxicologists traditionally use base 10 logarithms to calculate
the PS48,54 (the convention adopted for this chapter), while engineers often
use natural logarithms.55,56
When the LCt50 is constant with respect to t, it is said to obey Haber’s
rule (Haber, 1924).57 However, Haber’s rule is more of an exception than a
true rule. Thus, a new term, toxic load (TL), has been developed and exten-
sively used in IH toxicology to account for time dependent toxicity.58,59 TL is
n
normally expressed as C t, with n being the TLE. The same statistical theory
used for dosages48 has also been adopted for use with TL.55,60 Thus, LTL50
refers to the TL that will produce lethality in 50% of exposed individuals.
LTLXX and LTL50 can be substituted for LCtXX and LCt50 in eqn (9.7). Some
studies report a PS (kTL) for the TL.55,61–63
The TL relationship is empirical, rather than theoretical.55,60 Thus, eqn (9.7)
(when being used with TL) needs to be empirically derived on an individual
toxicant basis from experiments where both C and t are varied.64 When n = 1,
the chemical follows Haber’s rule.59 For the acute IH of irritant gases, n tends
to be greater than unity and is often of the order of two in value.58 The value for
n may offer some insight into the influence of underlying toxicological mecha-
nisms.60 For instance, it can be argued that if n > 1, then significant detoxifica-
tion may be occurring during the exposure, since the required effective dosage
is increasing as the dosage is incurred over a longer duration.
9.2.3 Probit Analysis Models Used for Fitting Response Data

For the present study, LCt50s were calculated from the experimental CK
lethality response data (even if the LCt50 values were originally reported),
which was collected from 16 sources. Whenever possible, PSs and TLEs
were also calculated (see Section 9.4.3). Parameter estimates were obtained
through the statistical analysis and modeling of experimental IH data. Tra-
ditionally, LCt50s are determined via the use of probit analysis.38 In conven-
tional probit analysis of binary response data (e.g. either the animal lives or
dies), two parameters are estimated simultaneously from the response data
using MLE:33,36 the median effective stress¶ and the standard deviations of
the effective stresses. The probit model can also be extended to multi-level
responses or an ordinal scale (e.g. mild, severe, lethal).33,37,41,42 A stable solu-
tion via probit analysis is dependent on several factors such as the number of
animals used, the number of exposure groups, dose spacing used, etc.18,38 If a
successful probit analysis is not possible,** then the median effective stress
can still be estimated via a one-factor MLE,†† 36 where the standard devia-
tion is kept at some fixed value (usually based on historical knowledge of the
problem) and an estimate is found for the median effective stress.
One of the following generic probit models (with or without some slight
modification thereof) was used for fitting experimental response data via
one-factor MLE/probit analysis/multifactor probit analysis/etc. (see Table
9.2).36,38,48,55 Either a binary or ordinal response (with a probit-link function)
¶
In toxicology, the median effective stress is the base 10 logarithm of the median effective dosage.
s
The PS equals the inverse of the standard deviation of effective stresses.
**This point is readily demonstrated if probit analysis is attempted and either: (a) there is fail-
ure to reach convergence on a stable solution for both PS and median lethal dosage estimates;
or (b) if convergence is achieved and the SEs associated with these estimates are ridiculously
large.
††
See either Hulet et al. (2006)37 or Sommerville et al. (2009)18 for an example of the one-factor
MLE analysis.
270 Chapter 9
was used with any of the equations in Table 9.2. With each equation, the
median effective dosage is determined from the final model fit by setting Z
equal to 0 and solving for log(C) or log(t). Both b’s and k’s are fitted coeffi-
cients (with the exception of the one-factor MLE, where some of the k’s are
kept constant), and the k’s correspond to PSs.
When fitting the equations in Table 9.2, all variability in the data will
contribute to the estimate for PSs (kC, kt and kTL), be it from variance due to
individual susceptibilities (the true goal), batch effects, experimental error,
differences in durations, compilation from many sources, etc.61 The heteroge-
neity introduced by outside sources of variance will artificially lower the PS,
which is why combining response data from multiple independent studies is
not recommended (as it introduces batch effects into the analysis).17,18,55,59,61
The precision of slope estimation typically improves as more experimental
subjects are used.
For studies where both C and t were varied, t was always treated as a factor
[eqn (9.9) and (9.11)] rather than as a covariate [eqn (9.12)] in order to produce
the best overall PS and median effective C or t estimates.37,42,59 This was because
any curvature in the log(C) versus log(t) relationship will artificially lower the
PS (insofar as the slope is a measure of the response variability among individ-
uals exposed to a toxicant). Eqn (9.12) was used only for calculating the TLE.
Table 9.2 Representative

probit models used to fit response data for the present
study.
Equation
Equation number Description
Z = b0 + kC log(C) or Z = b0 + kT log(t) [9.8] One-factor MLE (kC or kT kept
constant) or traditional
probit analysis (nothing kept
constant)
=Z kC log ( C ) + ∑ bT,i ti [9.9] Probit analysis with C as a
i covariate and t as a multi-
level factor [produces multiple
LCt50s and one probit slope
(kC)]
=Z kT log ( t ) + ∑ bC ,i Ci [9.10] Probit analysis with t as a
i covariate and C as a multi-
level factor [produces mul-
tiple LCt50s and one probit
slope (kT)]
=Z kC log ( C ) + ∑∑ bi , j ti Species j [9.11] Probit analysis with C as a
i j covariate and Species and t as
multi-level factors [eqn (9.11)
can be expanded to include
additional factors; produces
multiple LCt50s and one
probit slope (kC)]
Z = b0 + kC log(C) + kt log(t) [9.12] Binary regression of covariates
C and t [produces a TLE (TLE
= kC/kT)]
Eqn (9.13) is one form of expressing the final human TL estimate. To use it,
three parameters must be defined: the TLE (n), the PS (concentration; kC) and
the TL constant (k50%). The k50% can be calculated using the known ECt50 (the
effective concentration–time to 50% of exposed individuals (used for sub-lethal
effects in place of the LCt50)) or LCt50 at some reference duration. All three
parameters are chemical/endpoint specific.
⎡⎛ n ⎞ ⎤
=
ETL XX or (C nt )XX
LTL XX L= ( k50% ) antilog ⎢⎜ ⎟ Z ⎥ (9.13)
⎣⎝ kC ⎠ ⎦
9.2.4 Statistical Treatment of Historical Data
Proper evaluation of historical data is critical. The studies were often done
in different decades, by different researchers in different laboratories. With
linear regression analysis, the use of weights technically permits assigning a
numerical value to the quality of a datum, but how does the analyst properly
estimate the values for weighting? A solution to this problem is the use of
one of many existing robust regression methods.34–36 In this chapter, the IRLS
method was used.34–36 The IRLS method does not involve making an a priori
decision on what weight values to use. Instead, through an iterative process,
the weight of a datum is calculated as some function of its residual from the
previous iterative step. Often, the standardized residual is used,34 but for this
study the studentized deleted residual was used instead. The studentized
deleted residual of an observation uses a predicted value from a regression
fit omitting the observation, and it is more sensitive to outliers than the stan-
dardized residual. Weights based on deleted residuals will be less than those
based on standardized residuals (all other factors being equal). Weights (W)
were calculated for the present chapter using the following equation:
2
Wi , j =
⎡1 + TRES2(i −1), j ⎤
⎣ ⎦
convergence criteria: (9.14)
( )
for each j : W(i + 1), j − Wi , j ≤ 1 × 10−6
where the counter i represents the ith iteration, and commences at 2 since the first
regression run is unweighted, j is the index for the jth observation, and TRESi,j is
the studentized deleted residual for the jth observation within the ith iteration.
Convergence was considered reached when, for each Wj successive iteration
(i and i + 1), estimates were produced that agreed to the sixth decimal place.
ealthy Subpopulation Versus General Population in

9.2.5 H
Toxicity Sensitivity
It is assumed that only healthy animals were used in the reviewed studies.
Consequently, when scaling from smaller mammals up to humans, the
derived estimate should correspond to a healthy human subpopulation,
272 Chapter 9
defined in this case as being healthy and fit enough for military service. How-
ever, this does not address the issue of the more vulnerable portions of the
general population. Several studies have attempted to account for such sensi-
tivity differences in different populations.63,65–67 For this study, it is assumed
that the sensitive subpopulation can be adequately modelled using eqn (9.7)
if a PS and median effective dose for the general (or whole) population were
used. The vulnerable subpopulation would be accounted for by using the
lower tail of the bell distribution. Thus, the key is to change the basis (from
a healthy subpopulation to the general population) of the PS and median
effective dose estimates that were derived using the mammalian data. In a
series of reports,50,68,69 the US Army Edgewood Chemical Biological Center
(ECBC) has developed a mathematical method for the conversion from one
population basis to another, with Crosier (2007)69 presenting the final ver-
sion of the method. The premise of the approach is that mathematically the
distribution of log(doses) for a healthy subpopulation is located completely
within the distribution formed by the general population (i.e. the smaller
bell curve is inside the larger bell curve). A major advantage of this method
is that it reduces the amount of subjectivity that has been used in the past in
accounting for population differences.
9.3 CK: Properties and Characteristics

CK is a colorless gas above 55 °F (12.8 °C). As one of the “cyanides” it is
among the most rapidly acting of all known poisons. Inhaled CK is quickly
distributed systemically and inhibits many enzyme systems, the most criti-
cal effect being disruption of cellular respiration in tissues. Tissues highly
dependent on oxidative metabolism for energy production, such as the cen-
tral nervous system and myocardium are immediately affected, resulting in
the characteristic loss of consciousness, respiratory arrest and, ultimately,
death.26 However, unlike AC, to which it is closely related, CK is intensely
irritating to eye and respiratory tissue.31,32 As an irritant, CK has a delayed
toxic effect similar to lung irritants such as chlorine and phosgene.26 Munro
et al. (1999)70 have reviewed the environmental fate and toxicity of CK, and
its chemistry has been reviewed by Kikilo et al. (2008).71 Its significant prop-
erties are listed in Table 9.3.31,32
CK was investigated as a potential CW agent during WWI by several nations
and was subsequently used on a limited basis. According to US sources,72 the
French were the only nation to use CK on the battlefield, although there are
unconfirmed reports that the Germans may have used CK against the Rus-
sians and the Austrians against the Italians. CK possesses several properties
that made it an attractive alternative over AC:26,72

●● CK is heavier and less volatile than AC, which gives CK greater persistence
●● CK and AC were found to be approximately equivalent in their toxicity
and their speed of action
●● CK is an IH and ocular irritant, whereas AC is not
●● CK has the ability to penetrate German gas masks of the WWI period

Table 9.3 Characteristics
and properties of cyanogen chloride (from NIOSH 31,32
).
“CK”, “CC”, chlorine cyanide, chlorocyanogen,

Synonyms chlorcyan, chlorure de cyanogène (French)
CAS registry number 506-77-4
Molecular formula N≡C–Cl
Molecular weight 61.47
Physical state Colorless gas at room temperature
Odor Lachrymatory and irritating
Vapor density 2.1 (air = 1)
Liquid density 1.202 g ml−1 at 10 °C; 1.222 g ml−1 at 0 °C
Melting/boiling point −6.9 °C/12.8 °C
Flash point Nonflammable
Vapor pressure 760 torr at 12.8 °C; 680 torr at 10 °C; 448 torr at 0 °C
Volatility 2.62 × 106 mg m−3 at 12.8 °C; 2.37 × 106 mg m−3 at
10 °C; 1.62 × 106 mg m−3 at 0 °C (calculated from
vapor pressure)
Solubility Soluble in water and common organic solvents
Hydrolysis CK will hydrolyze in water with a half-life of 180
hours at ambient temperatures and pH 7.
Products are hydrogen chloride and cyanic acid
(CNOH)
Conversion factor (at 25 °C) 1 ppm = 2.51 mg m−3; 1 mg m−3 = 0.398 ppm
The French did not use pure CK (“Mauginite”) in their CW agent shells.
Instead, they used a mixture of 70% CK and 30% arsenic trichloride in their
munitions, which they called vitrite.72 It appears that arsenic trichloride may
have been added as a stabilizer by the French. After WWI, despite its limited
use in that conflict, CK was still part of the US CW agent stockpile up until
just after WWII,24 possibly due to its ability to penetrate some of the gas mask
canister materials of the era.28 An excellent review of US gas masks (research,
development, testing, canister design, etc.) from the WWI and WWII eras,
and the issue of CK penetration (and how it was eventually resolved) is pre-
sented in Volume 1 of Pierce (1946).73 During this period, CK was ranked
just below AC (the agent with the highest ranking) for its effectiveness as a
quick-acting non-persistent agent, and a non-persistent agent role were envi-
sioned for these two agents by the Allies if they were used in WWII.74 CK and
AC eventually were dropped from the US inventory as the ramifications of
the German discovery of nerve agents in the 1930s were fully appreciated in
the USA after WWII. Sarin (GB) replaced these agents in the inventory, taking
over the non-persistent agent role.24 However, even though it is no longer
weaponized by the USA, CK is still on the roster of chemicals for which the
performances of filter media are evaluated against.75
9.4 CK IH Toxicology

A substantial database exists of acute mammalian IH studies from military spon-
sored research (described below), but from open literature sources, there are
no known studies available. Military research on CK toxicology had essentially
274 Chapter 9
ceased around 1945 (see Section 9.4.3), and a summary of this research by Moore
and Gates (1946)28 has been made available publicly. However, much of the
underlying data summarized by Moore and Gates were not immediately released
to the public, and when large portions of the original US research were subse-
quently made available to the public (in a very low-key fashion), this event was
not noted by outside researchers. Thus, the perception of the toxicology commu-
nity at large is that very little experimental work is available on CK toxicology.29
9.4.1 General Toxicology

The target organs of acute CK exposure are the eyes and respiratory system
(extreme irritation) and the central nervous, respiratory and cardiovascular
systems (systemic effects). Like AC, CK inactivates the cytochrome oxidase
system, thus preventing cellular respiration and the normal transfer of oxy-
gen from the blood to body tissues. Exposure at high concentrations causes
effects within seconds and death within minutes in unprotected individuals.26
The biochemistry of AC and CK are similar, and the toxicity data for AC
is customarily extrapolated to CK. For example, Aldridge and Lovatt Evans
(1946)76 showed that the physiological effects of CK are similar to those of AC,
and demonstrated that CK is converted to cyanide in vivo. The study showed
that, except for the irritation of the respiratory passages, the physiological
response to CK can be explained by its conversion to cyanide in the body.
Cyanide could be detected in the circulating blood immediately after CK IH.
However, quite unlike AC, CK is a potent respiratory and ocular irritant.77
Its odor and effect are described as acrid, choking and lacrimating.78–80 Like
hydrogen sulfide (H2S), which shares the same systemic toxicity mechanism
as well as being a direct irritant, CK’s toxicity profile may be altered by the
“dual” attack of the local and systemic tissues.
9.4.2 Acute Human Toxicity

No reports are available that describe acute human lethal or severe effect
exposures to CK under known exposure conditions (i.e. concentration and
duration).81 For data on sublethal effects in healthy humans, there are two
primary WWII sources: Horton et al. (1943)80 and Anderson (1944).82
Horton et al. (1943)80 exposed five groups of five to seven volunteers to sep-
arate concentrations of CK ranging from 6.1 to 97.6 mg m−3 and determined
the following 3 min 100% effective concentrations (EC100s):

●● Eye irritation = 12.2 mg m−3
●● Tear formation (lacrimation) = 24.4 mg m−3
●● Tear overflow = 48.8 mg m−3

The authors concluded that CK was detectable by irritation of the eyes within
3 min at concentrations much less than those considered lethal for humans.
82
Anderson (1944) performed a series of human volunteer tests (six male
soldiers per group) using a 100 m3 chamber, and reported that in humans CK
is first detectable by its lachrymator effect between 0.54 and 2.6 mg m−3. At 17
mg m−3 painful blepharospasm is produced, and concentrations greater than
400 mg m−3 are judged to be immediately intolerable due to respiratory irrita-
tion. Based on the results of Anderson, Harrison (1944)83 concluded that the
harassing concentration for offensive purposes may be taken as 20 mg m−3.
9.4.3 Acute Mammalian Toxicity

There is a substantial body of government literature for CK. Toxicity studies
have been driven by its potential as a war gas. While these data are no lon-
ger classified, they are not generally available. Accordingly, those studies are
summarized below and relevant data are presented for the general audience.
For this chapter, a total of 89 individual median lethal IH dosages (LCt50)
and 16 PSs were found and/or calculated (see Section 9.6).‡‡ Every attempt
was made to identify and remove duplicate data points.§§ Data from anesthe-
tized animals were not used (when known). The following is a breakdown of
this database, collected or calculated¶¶ from 16 studies/sources dating back
to WWI:

●● From WWI: 51 LCt50 and 6 PS values
●● From WWII: 38 LCt50 and 10 PS values
●● Eight mammalian species represented (with number shown in brack-
ets): mouse (20), rat (12), guinea pig (13), rabbit (11), cat (5), monkey (7),
dog (14) and goat (7)
●● By source nation: USA (55) and UK (34)

This database was divided into four groups: USA during WWI (Table 9.4);84–89
UK during WWI (Table 9.5);90,91 US during WWII (Table 9.6);92–100 and UK
during WWII (Table 9.7).101 One study, Armstrong et al. (1933),92 did not fall
neatly into any of these four categories, but it was judged to be closer in char-
acter to the WWII period and so was placed into the US WWII group. In each
of these tables, the exact statistical methods that were used for estimating
LCt50s and/or PSs from the response data for each study are identified, along
with the number of animals and exposure groups used.
In addition to the UK and US research, toxicity data were also reported by
Flury and Zernick (1931).102 However, their data were not used in the present
‡‡
hen MLE was used to calculate the LCt50, a PS of 10 was assumed, and to calculate a LTL50, a
W
TL exponent value of 1.5 was assumed.
§§
This was particularly a problem for studies where there were numerous progress reports before
the publication of the final report.
¶¶
In some cases, LCt50s and PSs were not originally reported and were subsequently calculated
in this study using the reported response data via MLE (LCt50s) or probit analysis (both LCt50s
and slopes).
Table 9.4 CK
mammalian median lethal dosages from US studies during the WWI era.e
276
LCt50 (mg LC50 (mg
Study Species Methodb PS PS SE Groups Total Time tVM min m−3) m−3) LD50 (mg) Weight
Kolls et al. Mouse MLE NA NA 4 8 10 0.310 2850 285.0 0.09 0.6232
(1917)84,85 6 18 10 0.310 3480 348.0 0.11 0.8478
Marshall Mouse Probit 5.87 1.36 8 30 7.5 0.233 4540 605.3 0.14 1.9042
and 8 30 30 0.930 11 090 369.7 0.34 0.8558
Miller 3 12 60 1.86 13 250 220.8 0.41 0.9231
(1918)86,a 3 12 120 3.72 9550 79.6 0.30 1.3647
2 9 180 5.58 16 290 90.5 0.50 1.2457
3 12 240 7.44 12 810 53.4 0.40 1.6515
Rat Probit 12.82 3.60 8 18 7.5 1.52 5730 764.0 1.16 1.4421
7 14 30 6.09 8170 272.3 1.66 1.1729
5 14 60 12.2 14 910 248.5 3.03 1.5747
3 6 120 24.4 9600 80.0 1.95 0.6967
4 8 240 48.7 23 210 96.7 4.71 1.5467
Guinea Probit 18.30 4.99 8 16 2.5 0.455 5520 2208.0 1.00 1.9810
pig 7 12 7.5 1.37 8650 1153.3 1.57 1.7967
7 13 15 2.73 11 900 793.3 2.17 1.3962
7 11 30 5.46 15 270 509.0 2.78 1.2955
2 3 60 10.9 27 470 457.8 5.00 0.6522
2 4 180 32.8 93 400c 518.9 17.00
Rabbit Probit 17.27 4.38 10 21 7.5 10.9 5930 790.7 8.62 1.3285
8 16 30 43.6 14 090 469.7 20.47 0.6119
6 12 60 87.2 27 030 450.5 39.27 0.3870
2 4 240 349 104750c 436.5 152.20
Dog Probit 13.33 2.85 7 14 2.5 10.6 2130 852.0 9.01 0.8420
2 8 3.5 14.8 4440 1268.6 18.78 1.2542
13 26 7.5 31.7 4370 582.7 18.49 1.9440
5 10 15 63.5 3800 253.3 16.07 0.8662
Chapter 9
16 21 30 127 5270 175.7 22.29 1.0718
5 10 60 254 7760 129.3 32.82 1.6134
3 7 120 508 16 570 138.1 70.09 0.8335
Miller and Monkey MLE NA NA 4 4 7.5 10.5 4870 649.3 6.82 1.2992
Allometric Modeling of Mammalian Cyanogen Chloride Inhalation Lethality

Gross
(1918)87
West Cat MLE NA NA 2 2 30 25.4 7790d 259.7 6.58 0.8377
(1919)88,89
a
ot all response data collected by this study are shown in this table because it was not possible to calculate LCt50s for those species/duration groups
N
where all the animals either died or stayed conscious and survived.
b
For the MLE method, the PS was kept constant at 10 (except when noted).
c
Found to be an outlier in fit of eqn (9.14) and was dropped from the final fit.
d
Geometric mean of Ct of 6845 (survived) and 8859 (died).
e
Groups: number of individual groups of animals used for this duration, Method: method used to calculate LCt50 from experimental response data, NA:
not applicable, Time: exposure duration in minutes, Total: total number of animals exposed, tVM: time × minute volume in liters (but for model fit was
converted to m3: 1000 l = 1 m3), Weight: final weight value used in IRLS analysis for allometric fit using eqn (9.8).
277
278
Table 9.5 CK
mammalian median lethal dosages from UK studies during the WWI era.a,c
LCt50 (mg LC50 (mg
Study Species Method PS PS SE Groups Total Time tVM min m−3) m−3) LD50 (mg) Weight
b
Old Por- Rat Ordinal 8.62 1.54 2 10 5 1.02 9550 1910.0 1.94 1.9920
ton Red 2 5 10 2.03 10 940 1094.0 2.22 1.8404
Book91 and 2 5 60 12.2 12 880 214.7 2.61 0.7541
Boycott Guinea 2 6 5 0.91 8910 1782.0 1.62 1.2615
(1918)90 pig 1 4 20 3.64 16 630 831.5 3.03 1.9513
Rabbit 2 6 5 7.27 6870 1374.0 9.98 1.9897
2 4 30 43.6 28 790 959.7 41.83 0.4557
Cat 2 6 5 4.23 5820 1164.0 4.92 0.6345
1 2 30 25.4 14 330 477.7 12.11 1.1319
2 4 60 50.7 15 390 256.5 13.00 0.8395
Monkey 1 4 5 7.00 9070 1814.0 12.70 1.7918
2 4 10 14.0 11 470 1147.0 16.06 1.7279
1 2 20 28.0 11 020 551.0 15.43 1.4722
2 4 30 42.0 15 390 513.0 21.55 1.9279
2 4 60 84.0 22 500 375.0 31.50 1.3055
Dog 1 2 60 254 10 880 181.3 46.02 1.1843
Goat 1 2 5 48.0 6890 1378.0 66.14 1.3264
1 4 10 96.0 8310 831.0 79.78 1.4379
2 4 60 576 22 450 374.2 215.52 0.6315
a
ot all of the response data collected in this study are shown in this table because it was not possible to calculate LCt50s for those species/duration groups
N
where all the animals either died or stayed conscious and survived.
b
Data were originally reported as number of animals that died, number of unconscious animals (survived) and number of animals that did not fall into the
other two categories. An ordinal regression analysis (with probit link function) was performed on the dataset with a three level ordinal scale. The ordinal
regression also found the ratio of ECt50 (severe) : LCt50 to equal 0.60 (95% CI: 0.50 to 0.71).
c
Groups: number of individual groups of animals used for this duration, Method: method used to calculate LCt50 from experimental response data, Time:
Chapter 9
exposure duration in minutes, Total: total number of animals exposed, tVM: time × minute volume in liters (but for model fit was converted to m3: 1000 l =
1 m3), Weight: final weight value used in IRLS analysis for allometric fit using eqn (9.8).
Table 9.6 CK
mammalian median lethal dosages from US studies during the WWII era.d

LCt50 (mg LC50 (mg
Armstrong Mouse Probit 7.96 0.86 15 290 10 0.310 6670 667.0 0.21 1.2732
(1933)92
Bass and Dog MLE NA NA 4 4 6 25.4 5190 865.0 21.95 1.1687
Tucker
(1943)93,a
Coon et al. Mouse Probit 9.40 0.83 8 160 1 0.0310 (♂)4590 4590.0 0.14 1.5431
(1945)94 5 100 2 0.0620 (♂)5120 2560.0 0.16 0.7625
10 200 10 0.310 (♂)8000 800.0 0.25 1.9495
9 180 30 0.930 (♂)13 430 447.7 0.42 1.5557
Rat Probit 5.60 1.29 6 35 1 0.203 12 750 12 750.0 2.59 0.4888
Guinea 7 35 1 0.182 16 510 16 510.0 3.00 0.4447
pig
Rabbit 5 10 1 1.45 13 130 13 130.0 c 19.08
Cat 5 10 1 0.845 5900 5900.0 4.99 1.8029
Monkey 10 20 1 1.40 4650 4650.0 6.51 1.8209
Dog Probit 9.67 1.88 12 24 1 4.23 3280 3280.0 13.87 1.5236
9 18 3 12.7 4100 1366.7 17.34 1.1262
13 26 10 42.3 5380 538.0 22.76 0.8995
7 14 30 127 5960 198.7 25.21 0.6103
Franklin Goat Probit 8.08 1.83 22 66 2 19.2 6860 3430.0 65.86 0.7472
et al.
(1945)95
Fuhr and Rat Probit 5.78 0.93 8 96 2 0.406 19 420 9710.0 3.94 0.4021
Krackow
(1944)96
Ginzler et al. Dog Probit NA NA 11 11 2 8.46 3890 1945.0 16.45 1.3473
(1945)100,b
(continued)
279
Table 9.6 (Continued)
280
LCt50 (mg LC50 (mg
Krackow Rabbit Probit 5.77 1.56 6 60 2 2.91 13 790 6895.0 20.04 0.4078
and Fuhr
(1944)97
McGrath Goat Probit 7.16 2.28 9 30 2 19.2 3680 1840.0 35.33 1.5508
et al.
(1944)98
Silver and Mouse Probit 10.24 0.98 9 180 2 0.0620 6100 3050.0 0.19 1.3096
McGrath 8 160 10 0.310 7560 756.0 0.23 1.7525
(1942)99 7 140 30 0.930 13 740 458.0 0.43 1.4651
a
In this study, the following durations were used: 3.75 min (1 dog—lived); 5 min (2 dogs—1/2 deaths); and 13.5 min (1 dog—died). MLE was performed on
log(TL) instead on log(Ct), with a TLE of 1.45 and a PS (concentration) of 10 assumed [or PS (TL) of 6.9]. The geometric mean (6 min) of the four durations
was used for the final duration value.
b
In this study, amyl nitrite was being investigated as a treatment for CK IH poisoning. Only the results from the untreated dogs are shown here. The geo-
metric mean (2 min) of the 11 durations (ranging from 1.5 to 2.75 min) was used for the final duration value. MLE was performed on log(TL) instead of
log(Ct), with a TLE of 1.45 and a PS (concentration) of 10 assumed [or PS (TL) of 6.9].
c
Found to be an outlier in fit of eqn (9.14) and was dropped from the final fit.
d
Groups: number of individual groups of animals used for this duration, Method: method used to calculate LCt50 from experimental response data, NA:
not applicable, Time: exposure duration in minutes, Total: total number of animals exposed, tVM: time × minute volume in liters (but for model fit was con-
verted to m3: 1000 l = 1 m3), Weight: final weight value used in IRLS analysis for allometric fit using eqn (9.8).
Chapter 9
Table 9.7 CK
mammalian median lethal dosages from UK studies during the WWII era.a,b
LCt50 (mg
Study Species Method PS PS SE Groups Total Time tVM min m−3) LC50 (mg m−3) LD50 (mg) Weight
Short et al. Mouse Probit 4.98 0.56 5 50 0.5 0.0155 3470 6940.0 0.11 1.7929
(1944)101 3 29 1 0.0310 4030 4030.0 0.12 1.9966
7 70 2 0.0620 3710 1855.0 0.12 1.0658
4 40 3 0.0930 4060 1353.3 0.13 0.9853
Rat 3 30 2 0.406 11 680 5840.0 2.37 0.7249
2 20 3 0.609 5200 1733.3 1.06 0.8181
Guinea 1 8 0.5 0.0910 4500 9000.0 0.82 1.4024
pig 1 8 1 0.182 6310 6310.0 1.15 1.8872
3 30 2 0.364 7060 3530.0 1.28 1.4554
2 20 3 0.546 6040 2013.3 1.1 0.7965
Rabbit 1 6 1 1.45 6820 6820.0 9.91 0.7139
3 24 2 2.91 10 520 5260.0 15.29 0.5381
2 16 3 4.36 5350 1783.3 7.77 1.8385
Goat 2 4 1 9.60 4960 4960.0 47.62 0.8920
3 6 3 28.8 7800 2600.0 74.88 0.7477
a
Not all response data collected by this study are shown in this table because it was not possible to calculate LCt50s for those species/duration groups where
all the animals either died or stayed conscious and survived.
b
Groups: number of individual groups of animals used for this duration, Method: method used to calculate LCt50 from experimental response data, Time:
exposure duration in minutes, Total: total number of animals exposed, tVM: time × minute volume in liters (but for model fit was converted to m3: 1000 l =
1 m3), Weight: final weight value used in IRLS analysis for allometric fit using eqn (9.8).
281
282 Chapter 9
report because LCt50s could not be readily calculated from their data via pro-
bit analysis or MLE calculations.
9.5 P
revious Human Lethality Estimates for CK IH
Toxicity
There are only two known median lethality estimates developed for CK IH,
which are described below.
9.5.1 UK: Porton (Unofficial)

The UK military estimate is based on data from Short et al. (1944),101 in par-
ticular the note by Harrison on this work.83 Harrison compared the in-house
(Porton) data for AC and CK, and concluded that AC is twice as toxic for nearly
all species tested as CK for 10 min exposure durations. This reference dura-
tion was chosen because the irritant properties of CK likely cause animals to
hold their breath at higher concentrations of shorter durations (0.5–3 min),
compared with the non-irritant AC. In the dog, the most sensitive species,
there is a potency ratio of 2.6 : 1 (AC : CK), and this is used as the point of
departure for the extrapolation to humans. With the human ratio of AC : CK
set to 2 : 1 (and assuming that the human AC LCt50 at 1 min equals 5500 mg
min m−3),ss 103–105 and keeping that potency ratio constant for shorter times,
the lethal concentration for offensive purposes is reported to be 22 000 mg m−3
(30 seconds) and 11 000 mg m−3 (1 min). The author suggests that concen-
trations that are half of the lethal concentration would be expected to cause
unconsciousness lasting for at least 15 min. The harassing concentration for
offensive purposes is given as 20 mg m−3. Haber’s rule (TLE = 1) is implicitly
assumed by Harrison.
9.5.2 UK: Health Safety Executive

The Health Safety Executive (HSE) of the UK produces estimates for Specified
Level of Toxicity (SLOT) and Significant Likelihood of Death (SLOD) values
for land use planning purposes and for exposures involving the general pop-
ulation.55,61 At the SLOT level, the HSE has some expectation that highly sus-
ceptible people may be killed. Thus, the SLOT is based on 1% lethality levels
(e.g. LC01, LCt01, LTL01) observed in mammals. The SLODs are based upon
reported 50% lethality levels (i.e. LC50, LCt50, LTL50) in mammals. The HSE
typically bases its SLOT and SLOD values on the mammalian dataset that
produces the lowest LCtXX values.
The basis for the HSE CK estimate is documented by Lomax (2004).106
Lomax based the estimate on the WWII era University of Chicago research94
ss
The UK AC human LCt50 estimate (developed for offensive operations) was likely based upon
the mammal with the greatest inhalation resistance to AC, the monkey, as determined by
Barcroft (1923, 1931).103,104
28
as reported by Moore and Gates (1946). The most sensitive mammal (i.e.
lowest LCt50) was found to be the mouse. This was noted by Lomax to be con-
sistent with the mouse results of Flury and Zernike (1931)102 and thus pro-
vides support for considering this LCt50 value to be a reliable estimate. The
SLOD was based on the mouse 0.5 min LCt50 of 3000 mg min m−3 from Moore
and Gates. In the absence of CK PS information, Lomax used the HSE default
approach 55*** of dividing the LCt50 by 4 (effectively using a PS of 3.9) to arrive
at an LCt01 estimate (750 mg min m−3 in this case), which subsequently was
used to set the SLOT value. For the time dependence of CK toxicity, Lomax
concluded that a TLE of 1 provided the best fit to the data reported by Moore
and Gates (see Table 4 in Chapter 2 of Moore and Gates).28
9.6 Data Analysis and Results

9.6.1 Data Reduction
9.6.1.1 Calculation and Collection of Nominal LD50s
The collected data were subjected to several calculation procedures prior to
use for the final model fit [as previously used by Sommerville et al. (2009,
2010)17,18]. First, both the PS (with respect to concentration) and the LCt50
were estimated via either probit analysis or one-factor MLE (with a probit-link
function; see Section 9.2). One-factor MLE was only used if a stable solution
could not be reached with probit analysis, which was the case with the data-
sets from five studies.84,85,87–89,93,100 Response data from different studies were
never combined together for probit analysis (see Section 9.2.3). Probit analy-
sis calculations were performed using the binary logistic regression routine
in MINITAB™33 and the SE of the PS was recorded. A spreadsheet program
was used to perform one-factor MLE calculations.37 The resulting LCt50s,
LC50s, LD50s [via eqn (9.3)] and PSs are listed in Tables 9.4–9.7
In the case of the dataset from Boycott (1918),90 it was possible to perform
an ordinal regression (with probit-link function) using a three level ordi-
nal scale (2 = death; 1 = unconscious (severe); and 0 = less than severe). The
regression found that the ratio of the ECt50 to LCt50 equals 0.60 (95% CI: 0.50
to 0.71). Similar ratio values were calculated for the present chapter from
a review of the rat AC IH response data reported by Hartzell et al. (1985)107
(average of 0.60) and Levin et al. (1987)108 (average of 0.63).
9.6.1.2 Dataset Management for Probit Analysis

The following dataset possibilities exist (for studies with multiple concentra-
tions per duration) with respect to probit analysis (applicable CK IH studies
cited for each):
***Their default approach for a PS is based on a review of reported LC50 : LC01 ratios from pesti-
cides and lung damaging gases. It was found that the ratios ranged from 1.5 to 4. The con-
servative approach for protecting the public would be to assume a ratio of 4 in the absence
of other data.
284 Chapter 9
92,95–98
●● One species and one duration
●● One species and multiple durations99
●● Multiple species and one duration (not encountered in known CK
studies)
●● Multiple species and multiple durations86,90,91,94,101

Typically, in this report, probit analysis was applied (when appropriate) to
the total database of an individual study, with species, duration and/or gen-
der being treated as factors (if multi-level). However, if sufficient response
data were available, the total study database was split into subsets, with pro-
bit analyses applied to each subset. This produced a PS estimate and LCt50(s)
for each data subset. In some instances involving multiple species, a probit
analysis was not possible for every species subset that resulted from the split
of a multispecies dataset. In such cases, the LCt50 estimates were based on
the probit analysis of the main dataset, while probit analyses were performed
on the viable subsets to obtain additional PS estimates.
9.6.2 PS Estimation

A total of 16 PSs were calculated from the CK database and are summarized
in Table 9.8, along with the associated SEs. In addition to the PSs, several fit
statistics were also calculated using the following equations:
⎡ kC ⎤
ZT = ⎢ ⎥ (9.15)
⎣ SEkC ⎦
⎡ log 10 ( kC ) ⎤
Z T ,log = ⎢ ⎥ (9.16)
⎣ SElog kC ⎦
⎡ SE kC ⎤ ⎡ 1 ⎤
SElog kC = ⎢ ⎥ = ⎢ Z log ( 10 ) ⎥ (9.17)
⎣ kC log e ( 10 ) ⎦ ⎣ T e ⎦
The weighted geometric mean was taken for the PSs in Table 9.8, using
ZT,log as the weight. A geometric mean was used since it has been found that
experimental PSs follow a log-normal distribution.††† A common approach
is to use (1/SE2) as the basis for the weights in a weighted mean.36,109 How-
ever, the SEs of PSs are correlated with the PSs, with the bias towards giving
lower slope estimates greater influence than is warranted. ZT or ZT,log are
better indicators of the precision of a PS value. The weighted geometric
mean was found to equal 8.99 with a SE of 0.84 (95% CI: 7.4 to 11.0). For a
final recommended human (healthy) PS estimate, it was noted††† that post-
WWII PSs are larger (with statistical significance) as a group than those
from WWII and before. None of the CK PSs are from post-WWII studies. It
†††
Sommerville (2011), unpublished Chemical Security Analysis Center (CSAC) review of 121
lethal IH PSs taken from seven chemicals (ammonia, arsine, chlorine, CK, AC, hydrogen sul-
fide and phosgene).
Table 9.8 Probit
slope values calculated directly from response data reported by previous CK mammalian lethality studies.
SE on log10(kC)
Study Species PS (kC) SE on kC (SEkC) ZT log10(kC) (SElog kC) ZT,log
Marshall and Miller (1918)86 Mouse 5.87 1.36 4.32 0.7686 0.1006 7.64
Rat 12.82 3.60 3.56 1.1079 0.1220 9.08
Guinea pig 18.30 4.99 3.67 1.2625 0.1184 10.66
Rabbit 17.27 4.38 3.94 1.2373 0.1101 11.23
Dog 13.33 2.85 4.68 1.1248 0.0929 12.11
Boycott (1918)90 Rat, guinea pig, cat, mon- 8.62 1.54 5.60 0.9355 0.0776 12.06
key, dog and goat
Armstrong (1933)92 Mouse 7.96 0.86 9.26 0.9009 0.0469 19.20
Silver and McGrath (1942)99 Mouse 10.24 0.98 10.45 1.0103 0.0416 24.31
Short et al. (1944)101 Mouse, rat, guinea pig, 4.98 0.56 8.89 0.6972 0.0488 14.28
rabbit and goat
Krackow and Fuhr (1944)97 Rabbit 5.77 1.56 3.70 0.7612 0.1174 6.48
Fuhr and Krackow (1944)96 Rat 5.78 0.93 6.22 0.7619 0.0699 10.90
McGrath et al. (1944)98 Goat 7.16 2.28 3.14 0.8549 0.1383 6.18
Coon et al. (1945)94 Rat, guinea pig, rabbit, 5.60 1.29 4.34 0.7482 0.1000 7.48
cat and monkey
Mouse 9.40 0.83 11.33 0.9731 0.0383 25.38
Dog 9.67 1.88 5.14 0.9854 0.0844 11.67
Franklin et al. (1945)95 Goat 8.08 1.83 4.42 0.9074 0.0984 9.23
Weighted mean PS (kC) 8.99 0.84 10.71
285
286 Chapter 9
is assumed that the larger PSs are due to better laboratory controls reduc-
ing experimental variability that artificially reduces the measured PSs.
Thus, the recommended CK PS was adjusted for this effect by increasing
the geometric mean of 8.99 to 12, based on what was observed in AC PSs as
a function of the historical period.
9.6.3 Linear Regression Analysis

The statistical software package MINITAB™33 was used to calculate the fol-
lowing model fit for eqn (9.6) using the IRLS method [weights calculated
using eqn (9.13)]:

log(LC50) = (1.261) + (0.429)log(M) + (−0.683)log(tVM)
+ (−0.100)USWWI + (−0.118)Mouse (9.18)

with USWWI being an indicator variable for US studies conducted during
WWI (equals 1 for US WWI data and −1 otherwise) and Mouse being an indi-
cator variable for mouse data (equals 1 for mouse data and −1 otherwise). No
other individual species or study groups (i.e. USWWII, UKWWI, UKWWII) were
found to be outliers via standard statistical techniques.109,110 The statistics
for both weighted and unweighted fit are listed in Table 9.9. A plot of the experi
mental LC50s and the model fit for a healthy human [eqn (9.18)] with mass
M = 70 kg and VM = 0.015 m3 (with associated 95% CIs) are shown in Figures
9.1 and 9.2 as a function of duration and a function of inhaled volume (tVM),
respectively. An expression for the LTL50 (or L(Cnt)50) as a function of M and VM
was obtained by rearranging eqn (9.18) [see eqn (9.19)]. After further substi-
tuting eqn (9.2) for VM (with VM in units of m3 min−1), eqn (9.20) was obtained
for the LTL50 as a function of just M (with TLE = 1.46—see Section 9.6.5).

log(LTL50) = (1.845) + (0.628)log(M) + (−0.146)USWWI
+ (0.173)Mouse − log(VM) (9.19)
log(LTL50) = (5.147) + (−0.181)log(M) + (−0.146)USWWI

+ (−0.173)Mouse (9.20)

LTL50 is plotted as a function of M [using eqn (9.20)] in Figure 9.3 (with

USWWI and Mouse set equal to −1). For comparison, the individual LTL50s
calculated from the individual LC50s (using a TLE of 1.46) are also shown
in the figure, as well as the estimated healthy human LTL50. The log(LTL50)s
from the USWWI group in Figure 9.3 were adjusted by adding (2)(0.146) to
each such value to permit a better evaluation of the model fit as a function of
M. In Figure 9.4, the data and allometric fit for a healthy human are expressed
on a LCt50 versus t basis.
For the final human estimate (healthy subpopulation), eqn (9.18)–(9.20)
are used with USWWI = Mouse = −1. The former assumes that post-WWI
research gives more accurate (perhaps due to improvements in technology)
Table 9.9 Linear
regression results for allometric modeling of CK mammalian
median lethal concentrations.a
SE p-Value for
Fit Predictor Coefficient coefficient T-statistic T-statistic VIF
With Constant 1.2614 0.0564 22.35 0.0000
weights log(M) 0.429 0.0294 14.61 0.0000 5.300
log(tVM) −0.6834 0.0238 −28.74 0.0000 3.100
USWWI −0.0996 0.0172 −5.81 0.0000 1.340
Mouse −0.1183 0.0275 −4.30 0.0000 2.920
Fit S 0.1439 TLE TLE 1.46
statistics R2 94.4% SE on TLE 0.051
R2 (adj) 94.2% LL on TLE 1.36
F-statistic 343.3 UL on TLE 1.56
No weights Constant 1.2447 0.0754 16.52 0.0000
log(M) 0.4267 0.0395 10.80 0.0000 5.290
log(tVM) −0.6926 0.0323 −21.46 0.0000 3.240
USWWI −0.105 0.0232 −4.52 0.0000 1.390
Mouse −0.1421 0.0369 −3.85 0.0000 2.750
Fit S 0.1745 TLE TLE 1.44
statistics R2 91.0% SE on TLE 0.067
R2 (adj) 90.6% LL on TLE 1.31
F-statistic 204.9 UL on TLE 1.58
a
Adj: adjusted; LL: lower limit (95% CI), S: square root of the mean square error (MSE) of the
fit, UL: upper limit (95% CI), VIF: variance inflation factor.

of experimental mammalian CK LC50s as a function of
exposure duration versus the allometrically derived human (healthy)
estimate.
288 Chapter 9

of experimental mammalian CK LC50s as a function of
inhaled air volume versus the allometrically derived human (healthy)
estimate.
LCt50 determinations, and the latter assumes that human CK toxicity does
not follow that of mouse toxicity. Why the mouse should prove to be an out-
lier from the observed allometric trend is unknown.
Three individual LC50s were found to be statistically significant outliers
(see notes in Tables 9.4 and 9.6), with standard residuals with absolute values
greater than 2.33, and they were dropped from the final model fit (although
they are still shown in Figures 9.1–9.4). Another important item of note is
the discovery that there is statistical confounding of the study period with
exposure duration. Most of the WWI studies involved longer durations than
were used in the WWII studies.
9.6.4 Allometric Scaling of Mammalian CK Lethality

From eqn (9.18), the LC50 scales to body mass (M) with a scaling exponent
(bLC50) as follows:

bLC50 = (0.429) + (−0.683)bV (9.21)

From eqn (9.2), bV = 0.809 with a SE of 0.010, and thus bLC50 = −0.124 with a
SE of 0.036. The 95% CI for bLC50 equals −0.054 to −0.194. This interval does
not overlap zero—so the LC50 is a statistically significant function of M. Also,

of experimental mammalian CK LTL50 boxplots as a func-
tion of species body mass versus the allometric lethality fit.

of experimental mammalian CK LCt50s as a function of
exposure duration versus the allometrically derived human (healthy)
estimate.
290 Chapter 9
this dependence is not only due to the mass dependence of VM (see Section
9.2.1). Thus, in general, the larger the mammal, the lower the LC50 will be
(although this is a weak dependence). If the LTL50 is used instead [see eqn
(9.20)], the mass scaling factor is (−0.181) with a SE of 0.033 [assuming that
VM scales to 0.809 from eqn (9.2)].
9.6.5 TLE and Time–Concentration Relationship

From eqn (9.18), the TLE equals (−1/0.683) or 1.46 with a SE of 0.051. For
comparison, a list of TLE values and associated SEs calculated directly from
experimental response data reviewed for the present chapter [using eqn
(9.12)] is shown in Table 9.10. The individual TLEs range from 1.23 to 4.67,
and they are all larger than 1 with statistical significance (i.e. CK is not a
Haber’s rule chemical). The weighted mean [using (1/SE2) as the weights] was
found to equal 1.43 (95% CI: 1.33 to 1.52).‡‡‡ 35 The two TLEs with the largest
weights are both from mouse exposures: Silver and McGrath (1942)99 and
Coon et al. (1945).94 The weighted mean of the individual TLEs is in excellent
agreement with that derived from the allometric fit of the LC50s [eqn (9.18)]—
1.46. For the final human TLE, 1.46 was rounded to the nearest 0.05 or 1.45,
which is a better reflection of the precision of the estimate.
Table 9.10 TLE

values calculated directly from response data reported by previous
CK mammalian lethality studies.
Range of
durations Weights
Study Species (minutes) TLE SE (1/SE2)
Boycott Rat, guinea pig, 5–60 1.68 0.13 59.17
(1918)90 rabbit, cat, mon-
key, dog, goat
Marshall Mouse 7.5–240 1.48 0.12 69.44
and Miller Rat 7.5–240 1.59 0.13 59.17
(1918)86 Guinea pig 2.5–180 2.60 0.34 8.65
Rabbit 7.5–240 4.67 0.72 1.93
Dog 2.5–240 1.89 0.20 25.00
Silver and Mouse 2–30 1.39 0.040 625.00
McGrath
(1942)99
Coon et al. Mouse 1–30 1.45 0.023 1890.36
(1945)94 Dog 1.23 0.053 356.00
Overall weighted average (95% CI of average) 1.43 (1.33 to 0.049
1.52)
‡‡‡
The CI has been corrected for the overdispersion in the individual variances (as estimated by
the squares of the sample SEs) according to the method outlined by Myers and Montgomery
(1995).35 The SE of the final weighted mean is adjusted by multiplying by the square root of
the calculated χ2 statistic.
9.6.6 Human Lethality Estimates for CK IH

From eqn (9.18), a human (healthy) LC50 at 2 min was calculated using M =
70 kg, VM = 0.015 m3 and USWWI = Mouse = −1. The LC50 was found to equal
2049 mg m−3 (95% CI: 1720 to 2430 mg m−3), with the corresponding LCt50
equal to 4098 mg min m−3 (95% CI: 3450 to 4870 mg min m−3). This was
rounded to 4100 mg min m−3 for the final human (healthy) estimate. Using a
PS of 12, the LTLXX can be expressed [using eqn (9.13)] as:
⎡⎛ n ⎞ ⎤
=
LTL XX L ( C 1.45=
t ) XX ( k50% ) antilog ⎢⎜ =
⎟Z⎥ (1.27 × 10 ) antilog [( 0.121) Z ]
5
⎣⎝ kC ⎠ ⎦
(9.22)
At 50% lethality, Z = 0, so the LTL50 = 1.27 × 105 [(mg m−3)1.45 − min].
The Crosier model (see Section 9.2.5) was used to convert the lethality esti-
mate from a healthy subpopulation to the general population. The Crosier
model inputs used by Sommerville et al. (2009, 2010)17,18 were used for CK.§§§
The general population PS was found to equal 8.9, which was rounded to 9.0
for the final value, and the 2 min LCt50 was found to equal 3251 mg min m−3,
which was rounded to 3300 mg min m−3 for the final value. Using a PS of 9,
the LTLXX can be expressed as:
=
LTL XX L ( C 1.45=
t ) XX ( 9.26 × 10 ) antilog [( 0.161) Z ]
4
(9.23)
At 50% lethality, Z = 0, so the LTL50 = 9.26 × 104 [(mg m−3)1.45 − min]. Both
lethality estimates are compared with previous estimates (see Section 9.5) in
Figure 9.5.
9.6.7 Human Severe Effect Estimates for CK IH

From Section 9.6.1.1, the ratio of the ECt50 (severe) to LCt50 for CK has been
found experimentally to equal 0.60. Thus, the 2 min ECt50 (severe) for a
healthy subpopulation equals 4100 × 0.6 or 2460 mg min m−3, which was
rounded to 2500 mg min m−3 for the final estimate. Severe effects (for the
purpose of this estimate) are defined as unconsciousness/collapse. This fol-
lows the approach of Short et al. (1944)101 (see Section 9.5.1) and medical
opinion.111 Since severe effects and lethality result from the same toxic mech-
anism, the lethality PS (12) was adopted for severe effects. Using a PS of 12,
the ETLXX (severe) can be expressed as:

ETLXX = E(C1.45t)XX = (6.19 × 104)antilog[(0.121)Z] (9.24)

At 50% severe effects, Z = 0, so the ETL50 = 6.19 × 104 [(mg m−3)1.45 − min].
§§§
The critical Crosier parameters of δT and εT equal 0.900 and 0.744, respectively, for a single
truncation subpopulation. This assumes that the healthy subpopulation comprises 30% of
the general population.
292
Chapter 9
of previous human CK median lethal and severe effect dosage estimates with estimates of the present work.
For the general population, the lethality PS (9) was adopted for severe
effects. Using the Crosier model (with the same inputs as were used for
lethality), the ECt50 (severe) was found to equal 1982 mg min m−3, which was
rounded to 2000 mg min m−3 for the final value. Using a PS of 9, the ETLXX
can be expressed as:

ETLXX = E(C1.45t)XX = (4.48 × 104)antilog[(0.161)Z] (9.25)

At 50% effects, Z = 0, so the ETL50 = 4.48 × 104 [(mg m−3)1.45 − min]. Both
severe effect estimates are compared with previous estimates (see Section
9.5) in Figure 9.5.
9.6.8 C
omparison of Estimates: Present Chapter with
Previous Work
For the healthy subpopulation (see Figure 9.5), the estimates from the pres-
ent chapter are substantially different from those previously reported by
Porton in the UK (see Section 9.5.1), being lower than the previous estimate
for durations less than 20 min as well as having differing TLEs (1.45 for the
present chapter versus 1 for Porton). The most likely explanation for the dif-
ference is that the Porton estimates were developed for offensive battlefield
use. Thus, their LCt50 estimate was meant to aid in the targeting of the most
resistant individuals of the healthy subpopulation in the shortest duration
possible.
It should also be noted that Porton originally developed their 0.5–1 min
LCt50 estimate by extrapolation from the available 10 min mammalian LCt50s
using Haber’s rule (TLE = 1) with no adjustment for mammalian to human
differences. Thus, if their human LCt50 estimate was considered a 10 min
LCt50 rather than a 0.5–1 min LCt50, it would compare more favorably with
the 10 min LCt50 estimate from the present work [11 000 mg min m−3 (Porton)
versus 6800 mg min m−3 (present work)].
For the general population, the HSE estimate (see Section 9.5.2) is in
agreement with that of the present chapter only at short durations (from
0.5 to 2 min). Because of differing TLE values (1 for HSE and 1.45 for the
present work), the two LCt50 versus time relationships quickly diverge as
the durations get longer. If HSE had access to the full CK IH database,
it is likely that it would not have arrived at a TLE of 1, in which case, the
present chapter estimate would probably be in better agreement with that
of HSE.
There are no known lethal or non-lethal human exposures (occupa-
tional or otherwise) above a Ct of 1000 mg min m−3 against which the
lethality estimate of the present report can be compared (see Section
9.4.2). Concentrations of 400 mg m−3 are known to be immediately intol-
erable, and assuming a maximum duration of 1 min would only produce a
Ct of 400 mg min m−3, this is still about a factor of 6–8 below the proposed
0.5–1 min LCt50 and the HSE estimate. Therefore, it can be concluded that
294 Chapter 9
the proposed estimates are not too low in comparison to known human
exposures.
9.6.9 Comparison of CK and AC Human Lethality Estimates

A human (healthy) lethality estimate for AC IH has been developed by the US
Army (2005):23 a LCt50 (at 2 min) of 2900 mg min m−3, a TLE of 1.85 and a PS
of 10. This estimate is based on the work of McNamara (1976).¶¶¶ 19 A compar-
ison of the CK lethality estimate of the present chapter with the US Army AC
estimate is shown in Figure 9.6. AC is more potent for short durations, but
the relative potency reverses for durations longer than 20 min.
McNamara had extensively reviewed the existing database of mammalian
AC LCt50s in order to develop a new human estimate, drawing from many
studies conducted under US CW defense research efforts. The resulting data-
base (Table 1 in McNamara19) included lethal Cts for exposures ranging from
0.5 to 30 min, with ten mammalian species represented. The final AC LCt50s
[which were slightly modified by the US Army (2005)23] are based on 4 × mean
mouse LCt50s (at five separate durations). McNamara noted that goats and
monkeys are considered the most resistant species (among those studied)

of human (healthy) lethality estimates for CK (present
work) and AC [US Army (2005)].
¶¶¶
The US Army fit is based on the IRLS analysis fit (performed by the present author) of individ-
ual estimated LC50s (at 0.5, 1, 3, 10 and 30 min) for healthy humans produced by McNamara.
to AC IH poisoning, and his human LCt50 estimates fall into the sensitivity
range of these two species (by design).sss Mouse LCt50s were used since these
were available at all durations of interest (0.5–30 min).
9.6.10 Comparison of CK and AC Mammalian Lethality

McNamara (1976)19 did not investigate the use of an allometric model for AC
lethality. However, he does provide the following breakdown of the relative
sensitivity to AC IH toxicity by species with respect to the animal LCt50 values
that he reports:

●● Resistant to AC: sheep, goat, pig, monkey and guinea pig
●● Sensitive to AC: mouse, rat, rabbit and dog

With the exception of the guinea pig, all of the resistant species are
medium to larger mammals, and with the exception of the dog, the sensitive
species are smaller mammals. This suggests that the allometric modelling
of AC lethality may be possible, with the LC50 increasing as a function of M
(opposite to that observed for CK).
Using the TL model (with a TLE of 1.85), the AC LCt50 data of McNamara
(his Table 1; 42 values listed) were converted into LTL50s for this chapter,
and the mean log(LTL50)s (or µAC,j) were calculated for each species j (except
the pig and sheep). The mean CK IH µCK,js from the present chapter were
also calculated (using a TLE of 1.46 for the TL). For each agent i, the mean
of the µi,js was calculated (µ̄ i). These were then used as reference points
(within each agent) and were subtracted from each µi,j (µi,j − µ̄ i or Δµi; see
Table 9.11 and Figure 9.7). The antilog of Δµi equals the ratio of the geomet-
ric mean LTL50s. The resulting differences permit a ready comparison of
relative sensitivity among the species for each agent. The relative ranks of
each species’ mean log(LTL50) are also listed in Table 9.11. For comparison,
values for a 70 kg human were also calculated using the estimates shown
in Figure 9.6.
As shown in Figure 9.7 and Table 9.11, the µACs tend to be further away
from the mean (as represented by Δµi = 0 in Figure 9.7) on average than the
µCKs. An analysis of variance (ANOVA) was performed on the absolute values
of Δµi, or |Δµi|. The CK mean |Δµi|, or |ΔµCK|̄, was found to be less than that
for AC with a slight statistical significance (p-values of 0.101). In practical
terms, this equates to there being less distance (expressed as ratios of species
sss
The dog was ruled out as the basis for a human estimate by McNamara based on the results
of the unorthodox experiment by Barcroft during WWI.103,104 He placed himself and a dog
together into an AC exposure chamber, and both were exposed to a nominal concentration
of 625 ppm (690 mg m−3) for 1.5 min (or a Ct of 1035 mg min m−3). Barcroft experienced only
very minor effects, while the dog collapsed unconscious, had convulsions and then appar-
ently ceased breathing. After being left overnight in the lab, the dog was found to be walking
about the next morning and showed no further symptoms.
296
Table 9.11 Tabulation
of species mean log(LTL50)s, the corresponding agent mean log(LTL50)s and their differences for CK and AC.
Agent (i) CKa ACb CK AC
c d
Species (j) Bodymass Number µi,j Δµi,j Antilog Δµi,je Number of µi,j c
Δµi,j d
Antilog Δµi,j LTL50 rankf
e
(M) of LTL50s
LTL50s
Mouse 0.0207 20 5.2560 −0.1693 0.68 12 5.3393 −0.4371 0.37 7 8
Rat 0.261 12 5.5183 0.0930 1.24 7 5.7265 −0.0499 0.89 3 4
Guinea 0.323 13 5.6975 0.2722 1.87 3 6.3650 0.5886 3.88 2 1
pig
Rabbit 2.77 11 5.7380 0.3127 2.05 4 5.5781 −0.1983 0.63 1 6
Cat 3.36 5 5.3318 −0.0935 0.81 3 5.6650 −0.1114 0.77 6 5
Monkey 4.27 7 5.4035 −0.0218 0.95 2 6.0840 0.3076 2.03 4 3
Dog 13 14 5.1046 −0.3207 0.48 5 5.3640 −0.4124 0.39 8 7
Goat 36.9 7 5.3526 −0.0727 0.85 4 6.0890 0.3126 2.05 5 2
µ̄ CK of µ̄ AC of
5.4253
Δ μCK = 0.1695g 5.7764
Δ μAC = 0.3022g
SE of 0.118 SE of 0.179
Man 70.0 From fith 5.1362 −0.2891 0.51 From fiti 6.1496 0.3732 2.36 7 to 8j 1 to 2j
a
LTL50s calculated from present work using a TLE of 1.46.
b
LTL50s calculated from LCt50 database of McNamara (1976)19 (his Table 1) using a TLE of 1.85.
c
µi,j equals the mean of individual log(LTL50)s for agent i and species j. For CK values from USWWI, log(LTL50) adjusted upward by adding 2 × 0.146 based
on model fit (eqn (9.19)).
d
Δµi,j = (µi,j − µ̄ i), µ̄ i equals the mean of all j individual µi,j values for agent i.
e
The antilog of Δµi,j equals the ratio of the mean LTL50,i,j and mean LTL50,i.
f
Rank of µi,j from highest (1) to lowest (8) within each chemical.
g
Δ μi is the mean of the absolute values of Δµi,j for agent i.
h
From fit of the present work (using the model fit TLE of 1.46 instead of the final recommended TLE of 1.45).
Chapter 9
i
From US Army (2005)23 estimate.
j
Location of relative rank of the human µi, compared with above mammalian ranks.
Figure 9.7 Difference

between several species mean log(LTL50)s and correspond-
ing agent mean log(LTL50)s for CK and AC.
mean LTL50s) between the more sensitive and resistant species for CK IH
lethality compared with AC. Other relevant observations include (based on
Δµi values relative to zero):

●● The dog is highly sensitive to both CK and AC.
●● The rat and cat have the same relative sensitivity to both CK and AC
(both being close to the mean).
●● The mouse is highly sensitive to AC but not as sensitive to CK.
●● Several AC resistant species (guinea pig, monkey and goat) are not as
resistant to CK.
●● The rabbit is CK resistant but AC sensitive.
●● For the human, the µCK is less than both µCK,goat and µ̄ CK, which is
expected from the observed general allometric trend for CK—the LTL50
decreases as M increases (see Section 9.6.4). The reverse is observed
with AC, which would support an allometric trend of increasing LTL50
with increasing M.
●● For CK, the mouse and the human have approximately equal sensitivi-
ties. This supports the HSE approach to a human estimate (see Section
9.5.2) of using the mouse as the basis for a SLOD derivation (although
not the time dependence used by HSE).
298 Chapter 9
9.7 Discussion and Conclusions

Three parameters must be defined in order to establish an IH TL toxicity esti-
mate: the TLE (n), the PS (concentration; kC) and the TL constant (k50%). All
three parameters are chemical/endpoint specific, see eqn (9.13).
In this chapter, for a human estimate, both multifactor probit analysis [eqn
(9.12) and Table 9.10] and linear regression analysis [and allometric model-
ling; eqn (9.18)] have been used to established TLE estimates for CK. The PS
(concentration) has been estimated from experimental PS values (Table 9.8).
Lastly, the k50% (which equals the ETL50 or LTL50) has been found to be depen-
dent on the species body mass (M), with a mass scaling factor of (−0.181) and
a SE of 0.033. Thus, in general, larger mammals are more sensitive to CK IH
poisoning than smaller mammals, with the mouse being an outlier on this
allometric trend (see Sections 9.6.3 and 9.6.4).
A common problem in the allometric modeling of IH toxicity has been how
to best statistically treat non-independent parameters (see Section 9.2.1).
Often, both regressor and predictor terms are dependent on the species body
mass (M). A solution has been developed [eqn (9.6)] that is both faithful to
the definition of a nominal IH dose [eqn (9.5)] and eliminates non-independent
parameters.
Estimates for both severe effects and lethality have been developed in this
chapter on both a healthy subpopulation and general population basis [using
the Crosier model (2007)].69 Previous to this chapter, no such estimates had
been developed based on the larger body of available mammalian CK IH data.
Lastly, the modeling results for CK lethality have been compared to a sim-
ilar acting and more common chemical, AC (see Sections 9.6.9 and 9.6.10).
Important differences in trends between the two agents have been identified:
time dependent lethality (differing TLE values); allometric scaling; and the
degree of variability among species LTL50s. These differences may be due in
part to the previously described nature of CK’s systemic toxicity mechanism
as well as being a direct irritant (see Section 9.4.1).
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94. J. M. Coon, G. J. Rotariu, D. VanHoesen, R. K. Cannan and E. M. K.
Geiling, Cyanogen chloride: special toxicity studies, OSRD-5001, Office of
Scientific Research and Development, Washington, DC, 28 April 1945,
unclassified.
95. R. C. Franklin, J. L. Wilding, W. Stone and R. T. Franklin, Study of
short interval exposures of goats to CG, CK, and AC, MRL(DPG)-3, Medi-
cal Research Laboratory, Dugway PG, Tooele, UT, 28 November 1945,
unclassified.
96. I. Fuhr and E. H. Krackow, Cyanogen chloride LC50 for rats: 2 minute
exposures, TRL-27, Toxicological Research Laboratory, CWS, Washing-
ton, DC, 12 April 1944, ADB967754.
97. E. H. Krackow and I. Fuhr, Cyanogen chloride LC50 for rabbits: 2 minute
exposures, TRL-33, Toxicological Research Laboratory, CWS, Washing-
ton, DC, 31 May 1944, ADB967782.
98. F. P. McGrath, H. A. Sober and S. D. Silver, Cyanogen chloride LC 50 for
goats: 2 minute exposures, TRLR-26, Chemical Corps Technical Com-
mand, Army Chemical Center, MD, 28 March 1944, ADB967799.
99. S. D. Silver and F. P. McGrath, Cyanogen chloride. Part 1. preparation. Part
2. toxicity: median lethal concentration for mice, EA-TR-341, War Depart-
ment, CWS, Edgewood Arsenal, MD, 30 January 1942, unclassified,
ADB957368.
100. A. M. Ginzler, O. Bodansky, R. L. Feguson, B. J. Jandorf and C. L. Boyers,
The pathology of CK [cyanogen chloride] by inhalation, Medical Division
Report No. 34, Medical Research Laboratory of the Medical Division,
Office of the Chief, CWS, Edgewood Arsenal, MD, 19 June 1945.
101. R. H. D. Short, W. N. Watt, W. Anderson and K. P. Harrison, The effects
of CC on experimental animals and on human Subjects, PR-2603, Military
Intelligence Division, Chemical Defence Experimental Establishment,
Porton Down, Great Britain, 10 March 1944, unclassified, WO 189/2511.
306 Chapter 9
102. F. Flury and F. Zernike, Chlorcyan, in Schädliche Gase, Dämpfe, Nebel,
Rauch- und Staubarten, Verlag Julius Springer, Berlin, FRG, 1931, pp.
410–411.
103. J. Barcroft, Report on Toxicity of Hydrocyanic in Animals, War Department,
Experimental Station, Great Britain, 10 December 1917 as reported by G.C.
Armstrong, A.R. Koontz and M.G. Witherspoon, MG, The toxicity of hydro-
cyanic acid gas on dogs, monkeys, mice, guinea pigs and rabbits, EAMRD-20,
War Department, CWS, Edgewood Arsenal, MD, 31 December 1923.
104. J. Barcroft, The toxicity of atmospheres containing hydrocyanic acid
gas, J. Hyg., 1931, 31(1), 1–34.
105. G. C. Armstrong, A. R. Koontz and M. G. Witherspoon, The toxicity of
hydrocyanic acid gas on dogs, monkeys, mice, guinea pigs and rabbits,
EAMRD-20, War Department, CWS, Edgewood Arsenal, MD, 31 Decem-
ber 1923.
106. R. Lomax, Cyanogen Chloride DTL Assessment, Internal Memorandum
referencing HSE Report MH04-06 to Richard Rowlands, Health and
Safety Executive, Bootle, Merseyside, UK, 16 September 2004.
107. G. E. Hartzell, D. N. Priest and W. G. Switzer, Modeling of toxicologi-
cal effects of fire gases: II. Mathematical modeling of intoxication of rats
by carbon monoxide and hydrogen cyanide, J. Fire Sci., 1985, 3, 115–128.
108. B. C. Levin, M. Paabo, J. L. Gurman and S. E. Harris, Effects of exposure
to single or multiple combinations of the predominant toxic gases and
low oxygen atmospheres produced in fires, Fundam. Appl. Toxicol., 1987,
9, 236–250.
109. J. Neter, W. Wasserman and M. Kutner, Applied Linear Statistical Models.
Richard D. Irwin, Inc., Homeland, IL, 1985.
110. N. Draper and H. Smith, Applied Regression Analysis, John Wiley & Sons,
New York, 2nd edn, 1985.
111. J. A. Chalela and W. T. Burnett, Chemical terrorism for the intensivist,
Mil. Med., 2012, 177(5), 495–500.
Subject Index
2-methyl GF, as nerve agent, 17–18 incapacitants, 20–22
inter-war years, 4–5
AC. See hydrogen cyanide (HCN) lung injurants (choking
accidental exposures, of CG, agents), 12–13
124–125 chlorine (Cl2), 12–13
acute toxicity diphosgene (DP), 13
of Lewisite, 62–65 perfluoroisobutene
of sulfur mustard, 36–44 ((CF3)2C=CF2), 13
allometric modeling phosgene (CG), 13
and IH toxicology, 266–268 in the Middle East, 7–8
and mammalian lethality, nerve agents, 16–19
288–290 cyclosarin (GF), 17–18
animal models, and toxicokinetics DFP, 16–17
of SM, 194–195 2-methyl GF, 17–18
sarin (GB), 17–18
blood agents, 13–14 soman (GD), 17–18
cyanogen chloride (CK), 14 tabun (GA), 16–17
hydrogen cyanide (HCN), V agents, 18
13–14 physicochemical properties
of, 9–12
cancers, of SM, 186–187 gaseous and liquid
carcinogenicity, of SM, 54–58 agents, 9–11
CG. See phosgene (CG) solid agents, 11–12
chemical warfare agents (CWAs) prior to 1914, 2
blood agents, 13–14 riot control agents (RCAs),
cyanogen chloride 19–20
(CK), 14 and terrorism, 8
hydrogen cyanide vesicants (blister agents),
(HCN), 13–14 14–16
Chemical Weapons Convention Lewisite
(CWC), 8–9 (CHCl=CHAsCl2), 16
classification of, 9 nitrogen mustards,
during cold war, 6–7 15–16
ease of production, 12 sulfur mustard (SM),
history of, 2–9 14–15
307
308 Subject Index
chemical warfare agents (CWAs) properties and characteristics,
(continued) 272–273
during World War I, 2–4 cyclosarin (GF), 17–18
chlorine, 3
irritants, 3 DFP, as nerve agent, 16–17
overview, 2 diphosgene (DP), 13
phosgene, 3 dose-response statistics, 268–269
sulfur mustard, 4 DP. See diphosgene (DP)
during World War II, 5–6
Chemical Weapons Convention GA. See tabun (GA)
(CWC), 8–9 gaseous and liquid agents, of
chlorine (Cl2) chemical warfare, 9–11
as lung injurant, 12–13 GB. See sarin (GB)
usage in World War I, 3 GD. See soman (GD)
choking agents. See lung injurants GF. See cyclosarin (GF)
(choking agents)
CK. See cyanogen chloride (CK) Haber’s law, 125–127
clinical latent phase, of CG, hazard characterization, of OP
121–122 nerve agents, 86–104
clinical oedema phase, of CG, acute effects of exposure,
122–123 87–88
cold war, and chemical warfare, 6–7 delayed and long term
cutaneous injury, and SM, 181–183 effects of, 101–102
CWAs. See chemical warfare agents effects of low level exposure,
(CWAs) 102–104
CWC. See Chemical Weapons historical toxicity studies,
Convention (CWC) 88–100
cyanogen chloride (CK), 14 HCN. See hydrogen cyanide (HCN)
data analysis and results, HD. See sulfur mustard (SM)
283–297 human exposure, to SM
allometric scaling of effects on eyes, 155–158
mammalian lethality, effects on skin, 158–173
288–290 analytical considerations,
data reduction, 283–284 165–166
linear regression analy- clothing, 166–167
sis, 286–288 effects of RH, 164–165
PS estimation, 284–286 and increased tempera-
toxic load exponent (TLE) ture, 163–164
and time–concentra- liquid, 167–173
tion relationship, 290 vapour, 160–163
IH toxicology, 273–282 overview, 154–155
human lethality estimates human lethality estimation
for, 282–283, 291 for AC, 294–295
human severe effect estimates for CK, 282–283, 291
estimates for, 291–293 estimates for OP, 104
mammalian lethality, and toxicokinetics of SM,
295–297 207–209
Subject Index 309
hydrogen cyanide (HCN), 13–14 long-term effects
human lethality estimates of OP nerve agents, 101–102
for, 294–295 of sulfur mustard (SM)
cancers, 186–187
IH toxicology. See inhalation (IH) cutaneous injury,
toxicology 181–183
incapacitating agents, 20–22 ocular injury, 183–185
inhalation (IH) toxicology overview, 179–181
and allometric modeling, pulmonary injury,
266–268 185–186
cyanogen chloride (CK), lung injurants (choking agents),
273–282 12–13
human lethality estimates chlorine (Cl2), 12–13
for, 282–283, 291 diphosgene (DP), 13
human severe effect perfluoroisobutene
estimates for, 291–293 ((CF3)2C=CF2), 13
mammalian lethality, phosgene (CG), 13
295–297
dose–response statistics, maximum likelihood estimation
268–269 (MLE), 265
inhalation toxicokinetics, metabolism and distribution,
of SM, 201–204 of SM, 32–36
initial reflex syndrome, 120–121 Middle East, chemical warfare
injury mechanisms, of CG, in, 7–8
128–130 MINITAB™, 265, 283, 286
inter-war years, and chemical MLE. See maximum likelihood
warfare, 4–5 estimation (MLE)
intravenous toxicokinetics, mustard gas. See sulfur
of SM, 195–200 mustard (SM)
IRLS. See iteratively reweighted least mutagenicity
squares (IRLS) of Lewisite, 67
irritants, in World War I, 3 of sulfur mustard, 52–54
irritation and corrosiveness,
of SM, 44–48 nerve agents, of chemical
iteratively reweighted least warfare, 16–19
squares (IRLS), 265 cyclosarin (GF), 17–18
DFP, 16–17
Lewisite (CHCl=CHAsCl2), 16 organophosphorus (OP)
acute toxicity, 62–65 hazard characterization
mutagenicity, 67 of, 86–104
ocular toxicity, 65–66 history, 83–86
overview, 60–61 human estimates of
repeated dose toxicity, 66 toxicity, 104
toxicity for reproduction, 67 overview, 81–82
toxicokinetics, 61–62 physicochemical proper-
linear regression analysis, ties, 82–83
286–288 uses, 86
310 Subject Index
nerve agents, of chemical warfare pharmacodynamic
(continued) parameters, 222,
sarin (GB), 17–18 251–253
soman (GD), 17–18 physiological parameters,
tabun (GA), 16–17 218–221
V agents, 18 routes, 217
nitrogen mustards, as blister sensitivity analyses,
agent, 15–16 223–231, 254–258
simulations, 224–225,
occupational exposures, 253–254
of CG, 124–125 structure, 215–218,
ocular injury 231–251
and Lewisite, 65–66 subcutaneous data, 223
and sulfur mustard physicochemical properties,
(SM), 183–185 82–83
odour, of CG, 119 uses, 86
organophosphorus (OP)
nerve agents pathophysiology, of CG, 119–123
hazard characterization PBPK-PD model. See pharmaco-
of, 86–104 kinetic–pharmacodynamic
acute effects of exposure, (PBPK-PD) model
87–88 percutaneous toxicokinetics,
delayed and long term of SM, 204–206
effects of, 101–102 perfluoroisobutene ((CF3)2C=CF2), 13
effects of low level pharmacokinetic–pharmacody-
exposure, 102–104 namic (PBPK-PD) model
historical toxicity studies, and OP nerve agents
88–100 chemical specific
history, 83–86 parameters, 222
human estimates of data for fitting, 224
toxicity, 104 dermal data, 223
overview, 81–82 dosing parameters, 222
and pharmacokinetic– endpoints, 218
pharmacodynamic intravenous data, 223
(PBPK-PD) model overview, 214–215
chemical specific parameterization and
parameters, 222 validation data,
data for fitting, 224 222–223
dermal data, 223 pharmacodynamic
dosing parameters, 222 parameters, 222,
endpoints, 218 251–253
intravenous data, 223 physiological parameters,
overview, 214–215 218–221
parameterization and routes, 217
validation data, sensitivity analyses,
222–223 223–231, 254–258
Subject Index 311
simulations, 224–225, RCAs. See riot control agents (RCAs)
253–254 repeated dose toxicity
structure, 215–218, of Lewisite, 66
231–251 of sulfur mustard, 49–52
subcutaneous data, 223 riot control agents (RCAs), 19–20
phosgene (CG)
future therapeutic options, sarin (GB), 17–18
141–146 scavengers, and sulfur mustard,
Haber’s law, 125–127 206–207
as lung injurant, 13 sensitisation, of SM, 48–49
mechanisms of injury, 128–130 skin effects, and SM exposure,
overview, 117–118 158–173
properties, 119–123 analytical considerations,
clinical latent phase, 165–166
121–122 clothing, 166–167
clinical oedema phase, effects of RH, 164–165
122–123 and increased temperature,
initial reflex syndrome, 163–164
120–121 liquid, 167–173
odour, 119 vapour, 160–163
pathophysiology, solid agents, of chemical
119–123 warfare, 11–12
and stem cells, 145 soman (GD), 17–18
therapeutic research stem cells, and phosgene, 145
approaches, 130–141 subcutaneous toxicokinetics,
large animal in vivo of SM, 200–201
studies, 136–141 sulfur mustard (SM), 14–15
small animal in vivo action mechanism, 30–32
studies, 134–136 alterations to DNA, 31–32
in vitro studies, 131–134 chemical reactivity, 30–31
tolerance, 127–128 acute toxicity, 36–44
usage history, 123–125 carcinogenicity, 54–58
occupational/accidental human exposure to
exposures, 124–125 effects on eyes, 155–158
warfare, 123–124 effects on skin, 158–173
usage in World War I, 3 overview, 154–155
physicochemical properties, of irritation and corrosiveness,
CWAs, 9–12 44–48
gaseous and liquid agents, long-term effects of
9–11 cancers, 186–187
solid agents, 11–12 cutaneous injury,
PS estimation, for CK, 284–286 181–183
pulmonary injury, and SM, 185–186 ocular injury, 183–185
overview, 179–181
quantitative structure–activity rela- pulmonary injury,
tionship (QSAR) techniques, 218 185–186
312 Subject Index
sulfur mustard (SM), 14–15 intravenous, 195–200
(continued) overview, 191–192
mutagenicity, 52–54 percutaneous, 204–206
repeated dose toxicity, 49–52 subcutaneous, 200–201
sensitisation, 48–49
toxicity for reproduction, 58–60 usage history, of CG, 123–125
toxicokinetics of, 32–36 occupational/accidental
analytical procedures, exposures, 124–125
193–194 warfare, 123–124
animal models, 194–195
in humans, 207–209 V agents, of chemical warfare, 18
influence of scavengers, vesicants (blister agents), 14–16.
206–207 See also specific types
inhalation, 201–204 Lewisite (CHCl=CHAsCl2), 16
intravenous, 195–200 acute toxicity, 62–65
overview, 191–192 mutagenicity, 67
percutaneous, 204–206 ocular toxicity, 65–66
subcutaneous, 200–201 overview, 60–61
usage in World War I, 4 repeated dose toxicity, 66
toxicity for reproduction,
tabun (GA), 16–17 67
Tammelin esters, 18 toxicokinetics, 61–62
terrorism, and chemical warfare, 8 nitrogen mustards, 15–16
therapeutic research approaches, of sulfur mustard (SM), 14–15
CG, 130–141 action mechanism, 30–32
small animal in vivo studies, carcinogenicity, 54–58
134–136 irritation and
in vitro studies, 131–134 corrosiveness, 44–48
in vivo studies metabolism and
large animal, 136–141 distribution, 32–36
small animal, 134–136 mutagenicity, 52–54
TLE. See toxic load exponent (TLE) repeated dose toxicity,
tolerance, of CG, 127–128 49–52
toxicity for reproduction for reproduction, 58–60
and Lewisite, 67 sensitisation, 48–49
and sulfur mustard, 58–60 toxicity, 36–44
toxic load exponent (TLE), 290 toxicokinetics, 32–36
toxicokinetics volatility, 11
of Lewisite, 61–62
of sulfur mustard, 32–36 warfare usage, of CG, 123–124
analytical procedures, World War I, 2–4
193–194 chlorine, 3
animal models, 194–195 irritants, 3
in humans, 207–209 overview, 2
influence of scavengers, phosgene, 3
206–207 sulfur mustard, 4
inhalation, 201–204 World War II, 5–6

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Chemical Warfare Toxicology

Volume 1: Fundamental Aspects

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Print ISBN: 978-1-84973-969-6

© The Royal Society of Chemistry 2016

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Published by The Royal Society of Chemistry,

Registered Charity Number 207890

For further information see our web site at www.rsc.org

The international community has undertaken great efforts to abandon

Issues in Toxicology No. 26

Over the years, many of the reports produced as accounts of Government

Issues in Toxicology No. 26

Volume 1: Fundamental Aspects

Chapter 1 Development, Historical Use and Properties of

Issues in Toxicology No. 26

Chapter 3 Toxicology of Organophosphorus Nerve Agents 81

4.1 Introduction 117

Chapter 5 Human Exposures to Sulfur Mustard 154

5.1 Introduction 154

Chapter 6 Long-Term Effects of the Chemical Warfare Agent

6.1 Introduction 179

7.1 Introduction 191

Chapter 8 Modeling Organophosphorus Chemical Warfare

8.1 Introduction 214

Chapter 9 Allometric Modeling of Mammalian Cyanogen

9.1 Introduction 264

Subject Index 307

Volume 2: Management of Poisoning

Chapter 1 Treatment of Nerve Agent Poisoning 1

Chapter 2 Nerve Agents: Catalytic Scavengers as an Alternative

Chapter 4 Mustard: Pathophysiology and Therapeutic

4.1 I ntroduction 120

Chapter 5 Clinical and Laboratory Diagnosis of Chemical

5.1 Introduction 157

Chapter 6 Verification of Exposure to Chemical Warfare Agents 179

6.1 Introduction 179

Chapter 7 The Impact of New Technologies on the Elucidation of

7.1 Introduction 219

Chapter 8 Chemical Defence Against Fentanyls 259

8.1 Introduction 259

Subject Index 314

Development, Historical Use

Issues in Toxicology No. 26

1.2 Brief History of CW

1.2.2 The 1914–18 War (WWI)

1.2.3 The Inter-War Years

1.2.4 The 1939–1945 War (WWII)

1.2.5 Post WWII and the Cold War Years

Major advances were made in defensive countermeasures during this period,

1.2.6 The Middle East

1.2.8 Chemical Weapons Convention

1.3.2 Physicochemical Properties

Table 1.2 Boiling

1.3.3 Ease of Production

1.4 Main Classes of Chemical Agents

1.4.1.2 Phosgene and Diphosgene

1.4.2 Blood Agents

1.4.3 Vesicants (Blister Agents)

Scheme 1.1 Structures

Chapter 1 Development, Historical Use and Properties of

Chapter 3 Toxicology of Organophosphorus Nerve Agents 81

4.1 Introduction 117

Chapter 5 Human Exposures to Sulfur Mustard 154

5.1 Introduction 154

Chapter 6 Long-Term Effects of the Chemical Warfare Agent

6.1 Introduction 179

7.1 Introduction 191

Chapter 8 Modeling Organophosphorus Chemical Warfare

8.1 Introduction 214

Chapter 9 Allometric Modeling of Mammalian Cyanogen

9.1 Introduction 264

Subject Index 307

Chapter 1 Treatment of Nerve Agent Poisoning 1

Chapter 2 Nerve Agents: Catalytic Scavengers as an Alternative

Chapter 4 Mustard: Pathophysiology and Therapeutic

4.1 I ntroduction 120

Chapter 5 Clinical and Laboratory Diagnosis of Chemical

5.1 Introduction 157

Chapter 6 Verification of Exposure to Chemical Warfare Agents 179

6.1 Introduction 179

Chapter 7 The Impact of New Technologies on the Elucidation of

7.1 Introduction 219

Chapter 8 Chemical Defence Against Fentanyls 259

8.1 Introduction 259

Subject Index 314

1.2 Brief History of CW

1.2.2 The 1914–18 War (WWI)

1.2.3 The Inter-War Years

1.2.4 The 1939–1945 War (WWII)

1.2.5 Post WWII and the Cold War Years

1.2.6 The Middle East

1.2.8 Chemical Weapons Convention

1.3.2 Physicochemical Properties

1.3.3 Ease of Production

1.4 Main Classes of Chemical Agents

1.4.1.2 Phosgene and Diphosgene

1.4.2 Blood Agents

1.4.3 Vesicants (Blister Agents)

1.4.4 Nerve Agents

1.4.4.2 Sarin, Soman, Cyclosarin and 2-Methyl GF

1.4.4.4 Other Nerve Agents

1.4.5 Riot Control Agents

1.4.7 Future Developments

2.2 Sulfur Mustard

2.2.1 Mechanism of Action

2.2.1.3 Alterations to Other Cellular Components and Processes

2.2.2 Toxicokinetics, Metabolism and Distribution

2.2.2.3 In vitro Studies

2.2.3 Acute Toxicity

2.2.4 Irritation and Corrosiveness

2.2.5.2 Studies and Exposures in Humans

2.2.6 Repeated Dose Toxicity

2.2.7.1 DNA Damage In vitro

2.2.7.2 Mutagenicity Studies In vitro

2.2.7.3 Mutagenicity Studies in Drosophila

2.2.7.4 In vivo Studies in Mammals

2.2.8.3 Conclusions for Carcinogenicity

2.2.9 Toxicity for Reproduction

2.2.10 Summary of SM Toxicology

2.3.2 Acute Toxicity

2.3.3 Ocular Toxicity

2.3.4 Repeated Dose Toxicity

2.3.6 Toxicity for Reproduction

2.3.7 Summary of L Toxicology

3.2 General Substance Information

3.3 Hazard Characterization of Nerve Agents

3.3.1 Acute Effects of Nerve Agent Exposure

3.3.2 Historical Nerve Agent Toxicity Studies