Lapplicazione di metodi alternativi nella

valutazione del rischio tossicologico

07 Aprile 2016

www.Eurofins.com

BIBLIOGRAPHY

Indice

Paradigm shift in toxicology

Before:
responses in animals
towards an approach that: tests for effects on whole organism or certain
organ systems "one endpoint-one test"
Now:
understanding of the molecular mechanisms of toxicant effects in human cells
and tissues and implementation of 3R and "last resort" principles gives
prominent role to non-animal methods and approaches
Result: approaches become more flexible, combination of several methods,
case-by-case design depending of properties of test substance,

Paradigm shift in toxicology

New concepts in toxicological evaluation:
AOP based testing approaches for development of new method
IATA = Integrated Approach to Testing and Assessment: integrate and weight all
relevant data and guide the targeted generation of new data where required
ITS = Integrated Testing Strategy (combinations of test batteries covering relevant
mechanistic steps and organized in a logical, hypothesis-driven decision
scheme)Differently from tiered approaches not only the information generated in
the last step for a decision are considered.
WoE = Weight of evidence: evidence-based approach involving an assessment of
the relative weights (quality of data, consistency of data, severity of effect,
regulatory requirments on specific endpoint) of different pieces of the available
information (strenght vs weakness)
Useful as it makes use of all availabe information, even from less reliable studies,
when in pool with other information.
It requires expert judgement.

Adverse outcome pathways approach

It is based on the concept that a toxicant, after reaching and interacting
with a biological target, initiates a cascade of events which may lead to an
adverse outcome at the organism or population level.

Adverse outcome pathways approach

A recently published OECD Guidance Document (OECD, GUIDANCE
DOCUMENT ON DEVELOPING AND ASSESSING ADVERSE
OUTCOME PATHWAYS, 2013) provides guidance on which pieces of
information are necessary to identify a biologically plausible sequence of
key events leading to an observed effect supported by robust
experimental observations and mechanistic data
As a results a AOP Knowledge DataBase is under development.
It will be the starting point to identify non-standard methods that can be
associated with key events in one or more AOPs.

Regulatory acceptance processes of

newly develpoed methods

ECVAM = European Union Reference Laboratory for alternatives to animal testing

PARERE = Preliminary Assessment of Regulatory Relevance
NETVAL = Network of Laboratories for the Validation of Alternative Methods

Regulatory acceptance processes of

newly develpoed methods
Development of new method: verification of reliability and relevance of a test
method
1)scientific purpose, mechanistic basis, definitive
protocols with SOPs and endpoint measurments
2)variability over time and operators, using the
same lab set-up
3)Repetition in a laboratory different from the
one which has developed
4)evaluation in 3/4 laboratories with large
number of test substances.
5)how well the test can predict the reference
standard
6) chemical classes and/or endpoint for which a
test can be applied
7) At the end of validation, define the
reference chemical for new validation updates

Hartung et al (2004). A modular approach to the ECVAM principles on test validity. Altern. Lab. Anim. 32, 467-472.

Regulatory acceptance processes of

newly develpoed methods
Regulatory acceptance:
Formal acceptance by regulatory authorities indicating that the test method may be
used to provide information to meet a specific sectorial legislation (e.g. REACH,
PPPR, BPR, CPR)
- OECD Test guidelines: data generated in the testing in OECD Member country in
accordance with OECD Test Guidelines and OECD GLPs shall be accepted in other
Member (Mutual Acceptance of Data)
-EU Test Methods Regulation (TMR - Regulation (EC) 440/2008)
Is the way to make TGs law.
Anyway it is a slow process (methods have to be translated in alle EU official
languages): for this reason is not suitable source for new alternative methods.
As a consequence ECHA provides more flexible and quickly updatable docs
(Guidance ).
Finally REACH may allows use of methods that have not yet reached regulatory
acceptance on a case-by-case basis.

Biological endpoint:
Topical toxicity

-Skin corrosion and irritation

-Phototox
-Eye corrosion and irritation
-Skin sensitisation
Systemic toxicity

-Acute toxicity
-Genotoxicity & Carcinogenicity
-Repeated dose toxicity
Toxicokinetics

Skin Corrosion & Skin irritation

Skin contributes to a
variety of physiological
functions. The
importance of its
barrier property
increase the
importance of specific
dermal toxicology
The acute interaction
of the skin with
xenobiots may lead to
toxicological
response, involving
epidermis and dermis

Skin Corrosion & Skin irritation

Corrosion and irritation are distinguished according to the severity and reversibility
of effects:
Skin corrosion concern destruction of the skin:
- erosion of the stratum corneum
- death of the skin tissue and irreversibility of effects
Skin irritation (Irritant Contact Dermatitis - ICD)
- symptoms of rubor, calor, dolor, oedema

Skin Corrosion & Skin irritation

Adverse Outcome Pathways
Skin corrosion

Distruction of the tissue, after reaction with proteins, lipids cellular plasma
membrane

Erosion and necrosis

Skin irritation
Is a more complex event and includes:

Dermal bioavailability (intrinsic physicochemical properties of chemicals (size,

net charge, lipophilicity, partition coefficient (logP)

Metabolism (Skin has innate metabolic capacity especially in the epidermal

layers and it is conceivable that substances may be transformed to more
reactive metabolites by skin tissue

Tissue trauma (release of inflammatory mediators)

Activation on inflammatory pathway (increase of blood flow, permeability of
vessels, migration of immune cells, stimulation of nerves)

Skin Corrosion & Skin irritation

In vivo Acute Dermal Irritation/Corrosion OECD TG 404
Animal species: albino rabbit
Application of the test chemical : a dose of 0.5 mL of liquid or 0.5 g of solid or
paste is applied to the test site in a single dose to the skin (approximately 6
cm2) , in cases in which direct application is not possible (e.g., liquids or some
pastes), the test chemical should first be applied to the gauze patch, which is
then applied to the skin) untreated skin areas of the test animal serve as the
control.
Exposure period: 4 h (observation after 3 min and 1hr for corrosion) with up to
14d of observation period (to check for reversibility)
Stepwise approach: test using one animal + additional two animals

Skin Corrosion & Skin irritation

Scoring system
Corrosion: visible necrosis through the epidermis and into the dermis, following the
application of a test substance for up to four hours. Corrosive reactions are
typified by ulcers, bleeding, bloody scabs, and, by the end of observation at 14
days, by discolouration due to blanching of the skin, complete areas of alopecia
and scars
Irritation is the production of reversible damage of the skin following the application
of a test substance for up to 4 hours

Skin Corrosion & Skin irritation

Advantages:
Evidence of inflammatory- related response (edema and redness) due to the
increased diameter of blood vessels and modification of permeability.
Limitations:

difference between rabbits and human skins (usually rabbits are more sensitive
-> overprediction?)

inter-individual (inter-animal) variability

grading of skin responses is subjective
colored substances are difficult to score due the visual inspection of the skin
redness

Skin Corrosion & Skin irritation

For determination of skin corrosion and irritation several In vitro methods have
been validated and reached regulatory acceptance,
SKIN CORROSION
- Transcutaneous Electrical Resistance Test (TER) OECD TG 430
- Reconstructed Human Epidermis (RHE) OECD TG 431
- Membrane Barrier Test Method (Corrositex) OECD TG 435
All these three methods are stand-alone methods, allowing to classify a chemicals
to Cat.1 directly, altough they are related to different corrosivity mechanisms
(tissue disruption , pH, cells viability)
SKIN IRRITATION
- RHE Test Method OECD TG 439

Skin Corrosion & Skin irritation

Transcutaneous Electrical Resistance Test
(TER) OECD TG 430
Test system: excised skin discs from rats
Endpoint: skin barrier breakdown and distruction lead
to reduction of the electrical resistance across the skin
Applicable on: liquids (aqueous or non-aqueous),
semi-solids, solids (soluble or insoluble in water)
Classification:
Cut off value= 5 K; corrosive if TER<5 and/or skin is
damaged (dye penetration)
Cat.1 vs not corrosive (does not allow the
subcategorisation of Category 1)
Limitations: use of excised animal skin

Skin Corrosion & Skin irritation

Membrane Barrier Test Method (Corrositex) OECD TG 435
Test system: in chemico, the system is composed of an artificial membrane
(proteinaceous macromolecular aqueous gel and a permeable supporting
membrane) and a Chemical Detection System (CDS) = indicator solution below
the membrane
Endpoint: pH based. It measures time it takes a chemical to penetrate through
the membrane barrier to the indicator solution.
Classification: Corrosive (subcategory 1A
1B and 1C) vs non corrosive
Limitations: Aqueous chemicals with a pH
in the range of 4.5 to 8.5; chemicals that do
not cause detectable colour change in
CDS.

Skin Corrosion & Skin irritation

Reconstructed Human Epidermis (RHE) OECD TG 431
Test system: reconstructed human epidermis (RhE), which in its overall design
closely mimics the histology and physiological properties of the epidermis
Endpoint: Chemicals penetrate the stratum corneum by diffusion or erosion, and
are cytotoxic to the cells in the underlying layers. Cell viability is measured by
the MTT assay immediately after 3 and 60 min of exposure.
Classification: Cat.1
(subcategory 1A vs 1B-1C) vs
non corrosive. See example
table.
Limitations: chemicals reacting
with MTT or absorbing light in
the same range as MTT.
Extreme pH formulations can be
over-predicted (use Corrositex
for confirmation)

Skin Corrosion & Skin irritation

Reconstructed Human Epidermis (RHE) OECD TG 439
Test system: based on the same reconstructed human epidermis (RhE) of
OECD 431
Endpoint: Chemicals penetrate the stratum corneum by diffusion and cause tissue
trauma (cytotoxic to the cells in the underlying layers). Cell viability is measured
by the MTT assay immediately after a fixed exposure time (15 to 60 min
depending on RhE model)
Classification: requiring classification (cat. 1 or 2, further test to resolve between
these categories) vs not requiring classification (cannot classify as cat. 3)
Limitations: chemicals reacting with MTT or absorbing light in the same range as
MTT

Skin Corrosion & Skin irritation

Testing strategy
Both positive and negative results from in vitro tests are accepted if data are the results of a IATA (no in vivo confirmation is necessary)

1 data existing on human (occupational

and consumer exposure
2 old animal data
3 TOXNET: phys-chem (pH
water/octanol partition oxidising
capacity size of molecule and dermal
toxicity (absorption sensitization)
4identify similar compounds
5Acidy/Alkalinity reserve (for extreme
pH)
6In vitro data (bottom up or top down)

Skin Corrosion & Skin irritation

Final step: classification (example of CLP classification)

Eye corrosion and irritation

Chemicals interaction with eyes
mainly involves the primary site
of contact:
Cornea: Multi-layers transparent
epithelium
Iris: is a thin, circular structure in
the eye, responsible for
controlling the diameter and size
of the pupil (stroma, muscle,
epithelium)
Conjunctiva: the inside of the
eyelids and covers the sclera
(white part of the eye). It is
composed of non-keratinized
epithelim

Eye corrosion and irritation

Adverse Outcome Pathways
Four mechanism have been identified:

Eye corrosion and irritation

In vivo Acute Eye Irritation/Corrosion OECD TG 405
Animal species: albino rabbit
Application of the test chemical : 0,1 ml (100mg) of test item is placed on
conjunctival sac
Exposure period: eyes are not washed for 24h
Stepwise approach: test using one animal + additional two animals
Scoring system: The degree of eye
irritation/corrosion is evaluated by
scoring lesion s of conjunctiva, cornea,
and iris, at specific intervals for a
period of up to 21d in order to measure
persistance
Classification No cat vs Eye irritation
cat 2 vs Eye corrosion cat 1
The score depend on both severity
and/or perstincency of the damage

Eye corrosion and irritation

What endpoint correlates with classification?
Elaborating historical in vivo data:
Iritis rarely drives classification on its own -> no need to address it directly
Conjunctiva chemosis rarely drives classification on its own -> no need to
address it directly
Corneal opacity and conjuctiva redness address the classification mostly
of cat.2 chemicals -> potential issue: majority of in vitro methods mimic
opacity
Persistence without severity in the first 1-3days address cat.1 classification
-> potential issue: majority of in vitro are focused on immediate effect

Eye corrosion and irritation

Many in vitro test
methods have been
developed and
evaluated, some of
them reached
regulatory acceptance.
In vitro alternatives
method can be
grouped in 4
categories:
Organotypic assays
(CAM Isolated
organs), Cell based,
Rhe, In chemico
(Irritection)

Eye corrosion and irritation

Organotypic assays
Bovine Corneal Opacity and Permeability (BCOP)
OECD TG 437
Test system: isolated bovine cornea
Endpoint measured: corneal opacity and permeability
Protocol: liquids (neat) and surfactants (10%) exposed
for 10 min plus 2 hours post-exposure incubation;
solids (20%) exposed for 4 hours without postexposure incubation
Classification: No Cat. Vs Cat. 1
IVIS = mean opacity value + (15 x mean permeability
OD490 value)
Limitations:
No Cat: High level of FPs
Cat. 1 high FNs for solid (but 46% FNs are chemical
that will be classified based on persistence without
severity)

Eye corrosion and irritation

Isolated Chicken Eye Test Metho (ICE) OECD TG 438
Test system: chicken eyes isolated
Endpoints measured: additional endpoint in respect to BCOP: corneal
thickness to identify swelling and morphological damage. Scoring system for
opacity and permeability.
Limitations:
Cat. 1 Hgh FN for solid (but 75% FNs are chemical that will be classified based
on persistence without severity)

Hens Egg Test on the Chorioallantoic Membrane (HET-CAM)

Test system: chorioallantoic memvrane of chicken eggs
Endpoint: coagulation, haemorrhage, lysis
Classification:
Cat. 1 time to develop effect in 5 min exposure
No cat. time to develop effect in 0.5, 2 5 min exposure
Only method to address conjunctival effects
Limitations: sticky materials
The test is not OECD validated

Eye corrosion and irritation

Cell based assay
Fluorescein Leakage (FL) OECD TG 460
Test system: monolyaer of epithelial cells
MDCK
Endpoint: permeability to fluorescein
Protocols: 1 min exposure to chemicals
followed by 30 min incubation with
fluorescein
Classification: Cat. 1 vs Cat.2-no cat.
Limitations: Only chemicals soluble or
that form stable suspension at 250 mg/ml
High FNs rate
Cytosensor Microphysiometer (OECD draft)
Monolayer of L929 fibroblast, measuring celllular methabolic rate instead of
permeability

Eye corrosion and irritation

Short Time Exposure - STE (OECD 491)
Test system: Monolayer of SIRC cells (rabbit cornea)
Endpoint: MTT cytotoxicity
Protocol: Chemicals exposed at 5% and 0.05% for 5 min
Greater than 80% of a solution dropped into the human eye is excreted within
one to two minutes.The STE test method attempts to approximate these
exposure times
Classification: Category 1 when both the 5% and 0.05% concentrations
result in a cell viability 70%. No Category when both 5% and 0.05%
concentrations result in > 70%.
Limitations: Cat. 1 high FNs Only soluble or stable suspension.

Eye corrosion and irritation

Reconsturcted epithelium
EpiOcular Eye Irritation Test (EIT) OECD TG 492
Test system: multi-layered epithelium reconstructed from
primary human epidermal keratinocytes
Endpoint: cytotoxicity (MTT)
Protocol: exposure time of 30 min (6h for solids)
Classification:The test chemical is identified as (No
Category) if the mean percent tissue viability after exposure
and post-exposure incubation is > 60%
No Cat vs requiring classification (cant resolve cat. 1vs cat.
2)

Eye corrosion and irritation

In chemico methods
Ocular Irritection
Test system: Macromolecular matrix that mimics the structure of the cornea
Endpoint: Turbidity (opacity) as a measure of denaturation and disruption of
corneal proteins
Classification: Cat 1 vs No cat.
still under ECVAM validation
Limitations: Not applicable io chemicals with pH < 4 or pH > 9, oils, waterinsoluble organic chemicals, and intensely coloured chemicals

Eye corrosion and irritation

Measuring persistance
Currently missing in all in vitro methods
Methods under development:

-EVEIT (Ex-Vivo Eye Irritation Test)

Uses excised rabbit cornea monitors full-thickness corneal recovery
(epithelium and stroma) over 3 days using non-invasive Tomography

-PorCORA (Porcine Cornea Ocular Reversibility Assay)

Uses excised porcine cornea; is similar to BCOP, but by culturing the excised
corneas for up to 3 weeks, the reversal of damage to the cornea can be
measured over tover 21 days by fluorescein stain retention (re-staining of the
same tissue over the course of the study)

Eye corrosion and irritation

OECD GUIDANCE DOCUMENT ON BCOP AND ICE TEST METHODS:

HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE
IRRITANTS
in vitro tissues are different from in vivo (absence of inflammatory response) However,
the depth of injury may predict the degree and duration of the injury.
May be useful in a WoE approach

Eye corrosion and irritation

Regulatory acceptance:
it is possible to meet this information requirement of Annex VII with the in
vitro methods
Registrants, may be able to do so using Weight of Evidence considerations
by using Annex XI 1.2 adaptation possibilities and without testing in animals.
However, an in vivo eye irritation test may still be necessary depending on
the assessment of the available information and outcomes of in vitro studies.
It is generally accepted that no single in vitro test will replace in vivo assay.

Eye corrosion and irritation

Eye corrosion and irritation

Top down approach explained:
First method able to identify cat.1 from the rest with a low number of FNs
(identify as many Cat. 1. as possible)
Assay with low FNs reates are
BCOP (14% of FNs)
CM (21%)
These are the best assay for top-down approach
Considering the rates of FNs we should be able to identify up to 85% of Cat.1 just
from this first step (Most of FNs are chemicals underpredicted because their
classification is based on persistence)
- If result is cat.1 is confirmed -> no further testing
- In case of negative result a second step is needed, able to distinguish no cat. vs
the rest with a low number of FP

Eye corrosion and irritation

Top down approach
explained:

Eye corrosion and irritation

Bottom up approach explained
First method able to identify No cat. with assay with low FPs rates (identify
as many No cat. as possible):
Assay with low FPs reates are:
ICE
Epiocular
STE
Ocular Irrection
These are the best assay for bottum up approach
Considering the rates of FPs (20-40%) we should be able to identify 60% 80% of No. Cat. just from this first step
In case of positive result a second step is needed, able to distinguish cat 1 vs
the rest with a low number of FP

Eye corrosion and irritation

Bottom up approach
explained

Eye corrosion and irritation

Tiered approach conclusions:
- Increased coverage in the detection of No Cat. and Cat. 1 and Histology
could lead to higher confidence in a default Cat. 2 classification in the last tier

combine at least 2 methods for Cat. 2 classification!

- Most methods can be used to identify both Cat. 1 and No Cat., thus
increasing efficiency of the strategy

Skin Corrosion & Skin irritation& Eye full

testing strategy
There is a correlation between skin and eye (and usually eyes are more sensitive)
Data on skin toxicolgy can be used to correctly set-up a testing strategy:

Skin Corrosion & Skin irritation & Eye:

full testing strategy
Final step: classification (example of CLP classification)
H318 causes serious eye damage
H319 causes serious eye irritation.
. burns and eye damage and it is used for irreversible
H314 causes severe skin
damage to both skin and (H314 labeling makes H318 redundant)
It frequently happens that H318 (causes serious eye damage) classifies a
substance in combination with H315 (causes skin irritation) . Thats because
eyes can be more sensitive than skin

Phototoxicity
Toxic response after initial exposure of skin and subsequent exposure to light
(or skin irradiation after systemic administration of a chemical substance)
If a chemical absorbs UV or visible light, it needs to be determined if it is likely
to cause adverse phototoxic effects when intended for human use.
UV/vis absorption spectrum of the test chemical must be determined
according to OECD Test Guideline 101.
If molar extinction/absorption coefficient is < 10 litre x mol-1 x cm-1 the
chemical is unlikely to be photoreactive

Phototoxicity
In vitro testing
3T3 NRU phototoxicity OECD TG 432
The assay ased on cytotoxicity value of chemical when exposed/not exposed
to a dose of simulated solar light.
Criteria for the choice of an appropriate light source must include the
requirement that the light source emits wavelengths absorbed by
the test chemical (absorption spectrum); the dose of light should be of the UVA
and visible regions (UVB is highly cytotoxic, Infrared will increase heating), at
the highest non-cytotoxic dose (e.g. in the validation study: 5 J/cm2 [UVA])
Endpoint: Photo-irritation factor (PIF) = IC50 (Irr-) / IC50 (Irr+)
If IC50 cannot be calculate the MPE (mean photo effect) is calculate, based on
the comparison of the complete concentration/response curves
a test substance with a PIF < 2 or an MPE < 0.1 predicts: no
phototoxicity. A PIF >2 and < 5 or an MPE > 0.1 and < 0.15 predicts:

Phototoxicity

EPIDERM phototixicty test

Skin sensitisation
Skin sensitisation is a term used to denote the regulatory hazards known as
human allergic contact dermatitis
Skin sensitisation is an immunological process that is described in two
phases the induction of sensitisation and the subsequent elicitation of the
immune reaction (Kimber et al., 2002a)..
-Non covalent reaction
-while non-covalent reactions with metals and Redox cycling have been
linked to skin sensitisation
-Covalent bonds
the majority of the research has focused on chemicals which can
form covalent bonds with thiol and/or primary amino groups present in skin
proteins

Skin sensitization
The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent
Binding to Proteins
The first phase of inductions includes a sequential set of events which are
described in this AOP
four events that are recognised as key ones.

Skin sensitization
Induction phase:
1.Allergen penetrates the epidermis (potential biotrasformation)
2.Reaction with proteins in the skin (formation of hapten-protein conjugate)
3.Dermal Denditric cells and Epidermal dendritic cells (Langherans cells)
process the conjugate directly or after interaction with keratinocyte
4.Denditric cells activation and migration out of the epidermis to lymph nodes
5.In lymph nodes naive T-cells interact with dendritic cells
6.Differentition of T-cells to allergan specific memory T-cells, which are
recirculating in the whole body

Skin sensitization
Challenge phase (after a subsequent esposition to same hapten)
1.Allergen penetrates the epidermis (potential biotrasformation)
2.Reaction with proteins in the skin (formation of hapten-protein conjugate)
3.Dermal Denditric cells and Epidermal dendritic cells (Langherans cells)
process the conjugate directly or after interaction with keratinocyte
4.Circulating allergan-specific T-cells are activated and secrete inflammatory
cytokines
5. Inflammatory reaction is triggered and induce local response (Allergic
contact dermatis)

Skin sensitization

Skin sensitization
LLNA OECD 429
The other TG (i.e. TG 406) utilises guinea pig tests, notably the guinea pig
maximisation test and the Buehler test (13). The LLNA provides advantages
over TG 406 (13) with regard to animal welfare.
The mouse is the species of choice for this test. The chemical is applied to
the dorsum of each ear for 3 consecutive days. At day 6 radioactive labeller is
injected and Lymph nodes is analyzed
proliferation of T cells is measured in local lymph nodes which is proportional
to the dose and to the potency of the applied allergen (quantitative
assesment
in vivo radioactive labelling to measure an increased number of proliferating
cells in the draining auricular lymph nodes
Protocol:
A minimum of four animals is used per dose group, with a minimum of three
concentrations (series such as 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%,
with the highest concentration maximises exposure while avoiding systemic

Skin sensitization

DPRA = Direct Peptide Reactivity Assay

in chemico methods (based on organic chemistry) todetect and quantify
chemical reactivity.
Test system: synthetic heptapeptides containing ether cysteine or lysine
These approaches are generally limited by the lack of metabolism and
oxidation capacity,

Skin sensitization
KCs are the predominant cells in the skin and play a role in the initiation of
the immune
response by releasing a wide range of pro-inflammatory mediators and
growth factors
antioxidant/electrophile response element ARE/EpRE-dependent pathway,
plays acentral role in cellular defence against oxidative and electrophilic
stress, is relevant to the skin sensitisation process (Kim et al., 2008).

Skin sensitization
H-Clat (OECD draft dec. 2015)
nternalisation of
the hapten-protein conjugate by immature DCs. During this process, DCs
undergo a process
of maturation in which they lose their antigen processing capacity and
acquire the capability
to present the allergen to nave T lymphocytes. DC maturation is typically
measured
experimentally by assessing the expression of co-stimulatory and intercellular
adhesion
molecules (e.g. CD40, CD54, CD83, CD86) on the surface of DCs

Skin sensitization
The ability to detect pre-haptens (i.e. chemicals requiring abiotic activation)
and/or pro-haptens (i.e. chemicals requiring enzyme-mediated activation)
remains a challenge, since these sensitisers
are not systematically detected by test methods that lack or have limited
metabolic and
oxidative capacity.
Although these methods have been shown to perform well when compared to
conventional animal tests (Local Lymph Node Assay, LLNA) and available
human data, concerns have been raised on the regulatory acceptability of
negative results since it was questioned whether these methods are able to
predict chemicals that need to be activated to act as sensitisers
UPDATE 2016!
The pre-haptens, constituting the vast majority of chemicals requiring
activation, were mostly correctly identified by both the in chemico and in
vitro cell-based assays whereas the pro-haptens, which represent a small
subset of sensitising chemicals, were identified correctly by at least one of
the cell-based assays.
It was concluded that negative in vitro data should be accepted unless there

Skin sensitization
Test strategy (inserire 2of3 or tiered approach)
ECVAM Thus, when using in vitro methods, an approach might be first to
conduct an assessment of KE1 (e.g. DPRA) and if this proved negative, then
in the absence of other data to support an argument that it is nonsensitising,
there should be a follow up with a cell-based assay. If both tests were
negative, the substance would not be regarded as sensitising.
To support the discrimination between skin sensitisers (i.e. UN GHS Category
1) and nonsensitisers in combination with other
complementary information (i.e. in the context of an IATA)
, none of these three non-animal methods, DPRA, KeratinoSensTM or hCLAT should be used alone but always be considered in combinations and/or
with other information in the context of integrated approaches such as Weight
of Evidence (WoE) or Testing and Assessment Strategies. This is especially
the case, when negative predictions are obtained
The TGs cannot be used on their own to subcategorise

Skin sensitization
Substances may be incorrectly predicted if they:
Have a high cytoxicity
Have a low solubility in aqueous media (cell cultures)
Are not stable at high pH (DPRA)
Are pre- or prohaptens
The strategy is not yet applicable
To determine the potency
To assess complex mixtures/substances such as polymers and
formulations

Systemic Toxicity
-Systemic toxicity:
Acute toxicity (oral dermal respiratory)
Genotoxicity & Carcinogenicity
Repeated dose ( seurat - skin)

Systemic Toxicity
-Acute toxicity
Acute systemic toxicity testing involves an assessment of the general toxic
effects of a single dose or multiple doses of a chemical or product, within 24
hours by a particular route (oral, dermal, inhalation), and that occur during a
subsequent 14-days observation period. The substance may be administered
orally, by inhalation or dermally.
Test method

OECDTest Guideline

Endpoint

Acute Oral Toxicity - Fixed Dose Procedure

TG 420

Acute Oral toxicity - Acute Toxic Class Method

TG 423

Evident
toxicity
Lethality

Acute Oral Toxicity: Up-and-Down Procedure

TG 425

Lethality

Acute Dermal Toxicity

Acute Inhalation Toxicity
Acute Inhalation Toxicity - Acute Toxic Class
Method

TG 402
TG 403
TG 436

Lethality
Lethality
Lethality

Refinement on Endpoint: Avoiding lethality -> evident signs of toxicity (only TG420). FDP Also for Inhalation and Dermal?
It is possible to waive the dermal toxicity test if not classified by the oral route (i.e. oral LD50 > 2000 mg/kg) agreed at CARACAL 15

Systemic Toxicity - Acute toxicity

-Acute toxicity

It is possible to waive the dermal toxicity test if not classified by the oral
route (i.e. oral LD50 > 2000 mg/kg) agreed at CARACAL 15

Systemic Toxicity - Acute toxicity

-Oral toxicity OECD 420
It is a principle of the method that in the main study only moderately
toxic doses are used, and that administration of doses that are
expected to be lethal should be avoided. Also, doses that are known to
cause marked pain and distress, due to corrosive or severely irritant
actions, need not be administered.
The starting dose level is selected on the basis of a sighting study as
the dose expected to produce some signs of toxicity without causing
severe toxic effects or mortality

Systemic Toxicity - Acute toxicity

Animals are observed individually after dosing at least once during the
first 30 minutes, periodically during the first 24 hours, with special
attention given during the first 4 hours, and daily thereafter, for a total
of 14 days
During the study animals were observed for 14 days for signs of
possible toxic symptoms including:
- mortality
- evaluation of body organ functions
- evaluation of tegumentary apparatus conditions
- evaluation of mucosae conditions
- evaluation of somatomotor activity and sensorium conditions
- Body weight weekly reported.
- Macroscopic and microscopic changes in autopsy

Systemic Toxicity - Acute toxicity

the majority of chemicals are acutely toxic to humans by basal cytotoxicity,
that is, by interfering with general cell functions common to all cells
Mechanisms common to many cell types leading to organ failure include, for
example, disruption of membrane structure or function, inhibition of
mitochondrial function, disturbance of protein turnover, and disruption of
metabolism and energy production.
OECD No. 129 GUIDANCE DOCUMENT ON USING CYTOTOXICITY
TESTS TO ESTIMATE STARTING DOSES FOR ACUTE ORAL SYSTEMIC
TOXICITY TESTS
The NRU in vitro basal cytotoxicity assay procedure is based on the ability of
viable cells to
incorporate and bind neutral red (NR), a supravital dye. Toxicants can alter
the cell surface or the lysosomal membrane to cause lysosomal fragility and
other adverse changes that gradually become irreversible.

Acute toxicity
Range finder test
This is the initial cytotoxicity test performed to determine the starting doses
for the main test. The NRU assays test eight concentrations of the test
substance by diluting the stock test substance solution in log dilutions to
cover a large concentration range .
Main test
The main test of the cytotoxicity assays is performed to determine the IC50
value. The concentration closest to the range finder test IC50 value serves as
the midpoint of the concentrations tested in the main test. Compared to the
range finder test, the main test uses a smaller dilution factor for the
concentrations tested

Acute toxicity
A linear regression-model using the log-transformed IC50 values and logtransformed rodent oral LD50 values was developed for the prediction of
acute oral LD50 values from IC50 values.
For substances with no molecular weight, IC50 values in g/mL can be used
in the following regression formula to estimate the LD50 in mg/kg:
log LD50 (mg/kg) = 0.372 log IC50 (g/mL) + 2.024

The limitations of the in vitro NRU methods are largely due to the differences
between whole animal and cell culture systems. Animal and cell culture
systems are different with respect to how a substance or toxicant is delivered
to the cell and how it is distributed within the cell and metabolized.
The toxicant is in direct contact with cells, leading high FPs results. Finally
information on tissue specific toxic effect are not present.

Systemic Toxicity - Acute toxicity

However, despite this good correlation, the NICEATM/EURL ECVAM
validation study found that the neutral red uptake (NRU) basal cytotoxicity
tests were not able to predict with sufficient accuracy
the acute oral toxicity categories as defined by the United Nations Globally
Harmonised System (UN GSH, 2011).
The peer-review of this study concluded that the NRU basal cytotoxicity test
methods, including the test performed with the 3T3 mouse embryonic
fibroblast cell line (3T3 NRU), may be useful in a weight-of-evidence
approach to determine the starting dose for acute oral in vivo toxicity tests

Systemic Toxicity - Repeated dose

toxicity
Repeated dose toxicity comprises the adverse general toxicological effects
occurring as a result of repeated daily dosing with, or exposure, to a
substance for a specified period up to the expected lifespan of the test
species (sub-acute, subchronic and chronic exposures, including 28 day
oral, dermal and inhalation studies in rodents, 90 day oral, dermal and
inhalation studies in rodents, 90 day oral study in non-rodents, and chronic
toxicity studies in rodents)
These tests provide information on possible adverse effects on target organs,
on dose-response relationships, and on the reversibility/irreversibility of the
effect
These in vivo tests generate the no-observed-adverse-effect level (NOAEL).
which is used in the calculation of the MoS (Margin of Safety) or MoE (Margin
of Exposure). Overall, there is limited knowledge of the underlying
mechanistic pathways

Systemic Toxicity - Repeated dose

toxicity
Test method

28-day Oral Toxicity

Study in Rodents
90-Day Oral Toxicity
Study in Rodents
90-Day Oral Toxicity
Study in Non-Rodents
Dermal Toxicity: 21/28day Study (rat, rabbit or
guinea pig)
Dermal Toxicity: 90-day
Study (rat, rabbit or
guinea pig)
Inhalation Toxicity: 28Day Study in Rodents
Inhalation Toxicity: 90day Study in Rodents
Chronic Toxicity Studies
in Rodents

COUNCIL REGULATION
(EC) No 440/2008

Test Method
B.7

OECDTest
Guideline

Endpoint

in vitro/in vivo

TG 407

Repeated dose

in vivo

B.26

TG 408

Repeated dose

in vivo

B.27

TG 409

Repeated dose

in vivo

B.9

TG 410

Repeated dose

in vivo

B.28

TG 411

Repeated dose

in vivo

B.8

TG 412

Repeated dose

in vivo

B.29

TG 413

Repeated dose

in vivo

B.30

TG 452

Repeated dose

in vivo

Systemic Toxicity - Repeated dose

toxicity
Currently there are not test methods under
validation

Genotoxicity
Genetic alterations in somatic and germ cells are associated with
serious health effects, which in
principle may occur even at low exposure levels.
Genotoxicity is a broader term and refers to processes which alter the
structure, information content or segregation of DNA and are not
necessarily associated with mutagenicity. Thus, tests for genotoxicity
include tests which provide an indication of induced damage to DNA
Anyway Genotoxicity tests are usually taken as indicators for
mutagenicity effects (Mutations in somatic cells may cause cancer )
Mutations in germ cells can lead to spontaneous abortions, infertility or
heritable damage to the offspring and possibly to the subsequent
generations.

Genotoxicity
Test Method

COUNCIL
OECD Test
REGULATIO Guideline*
N (EC) No
440/2008
Test
Method

endpoint

In vitro/in vivo

Bacterial reverse
mutation test
(Ames test)

B.13-14

TG 471

Gene mutations

vitro

In vitromammalian
B.10
chromosome aberration
test

Updated TG 473

Structural
aberrations

vitro

Mammalian cell gene

mutation test
In vitromammalian cell
micronucleus test

B.17

TG 476 (under
revision)
Updated TG 487

Gene mutations

vitro

Structural and
numerical
aberrations

vitro

Mammalian erythrocyte
micronucleus test

B.12

Updated TG 474

Structural and
numerical
aberrations

vivo

Mammalian bone
marrow chromosome
aberration test

B.11

Updated TG 475

Structural
aberrations

vivo

Transgenic rodent
somatic and germ cell
gene mutationassays

TG 488

Gene mutations

vivo

In vivomammalian
alkaline cometassay

TG

DNA damage

vivo

Genotoxicity
Testing strategy: Combinations of in vitro genotoxicity tests and in
vivo follow up studies (EFSA approach)
For an adequate evaluation of the genotoxic potential of a chemical
substance, different end-points:
-Gene mutation
-Chromosomal aberrations (structural)
-Chromosomal aberrations (numerical)
An adequate coverage of all the above-mentioned end-points can only
be obtained by the use of multiple test systems (i.e. a test battery), as
no individual test can simultaneously provide information on all endpoints.

Genotoxicity

the majority recommend use of a basic test battery, comprising two or

more in vitro tests, or in vitro tests plus an in vivo test, to evaluate
genotoxic potential.
The bacterial reverse mutation test is always accepted as part of every
strategy because of its specificity for detection of genotoxic
carcinogens and is usually the first test to be performed. However, a
bacterial mutation test is unable to detect genotoxic substances which
target chromosomal integrity or segregation, or which affect genomic
stability by indirect mechanisms, for example by disturbance of DNA
repair fidelity, cell cycle control or apoptosis
As mentioned earlier, the in vivo follow-up test needs to be a logical
choice, i.e. the test should cover the same genotoxic endpoint as the
one which showed positive results in vitro.
As a follow-up for in vitro positives for clastogenicity or aneugenicity,
the in vivo mammalian erythrocyte micronucleus test is suitable. As a
follow-up for in vitro gene mutation positives, both the transgenic
rodent gene mutation assay and the Comet assay are suitable.

Genotoxicity
If all in vitro endpoints are clearly negative in adequately conducted tests, then it
can be concluded with reasonable certainty that the substance has no genotoxic
potential. Anyway some genotoxic compounds are still detected in vivo but not in
vitro
In addition the high false positive rate of the established in vitro genotoxicity tests
leads to an increased number of follow up in vivo tests needed for the confirmation
of these results.

In situations where there is exposure to very low concentrations of substances in

food/feed, an alternative approach, the Threshold of Toxicological Concern (TTC)
has been proposed. Application of the TTC approach requires knowledge only of
the chemical structure of the substance concerned and reliable information on
human exposure.

Genotoxicity

Genotoxicity
Most commonly used in vitro methods
The most commonly used methods for assessing the genotoxic
potential of substances are listed below, together with the relevant
OECD Test Guideline (TG) on the basis of their principal genetic endpoint:
Studies to investigate gene (point) mutation:
Bacterial reverse mutation test in Salmonella typhimurium and
Escherichia coli Ames test (OECD TG 471)
In vitro mammalian cell gene mutation test (OECD TG 476)
Studies to investigate chromosome aberrations:
In vitro mammalian chromosomal aberration test (OECD 473)
In vitro mammalian cell micronucleus test (OECD TG 487)

Genotoxicity
Studies to investigate gene (point) mutation:
Bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli
Ames test (OECD TG 471)
Amino-acid requiring strains can reverse their mutation and acquire the ability to
grow in the absence of the required a.a.
Limitations are the Test system (prokaryotic cells which is different from
mammalian cells in terms of metabolism, updatake, chromosome structure) . Also
it can reveal oonly direct damage and not indirect mechanisms (responses to DNA
damage, cell cycle control and apoptosis.)
In vitro mammalian cell gene mutation test (OECD TG 476)
L5178Y mouse lymphoma cells are exposed to toxicant measure mutation
thymidine kinase (tk) and hypoxanthine-guanine phosphoribosyl transferase (hprt)
loci

Genotoxicity

Studies to investigate chromosome aberrations:

In vitro mammalian chromosomal aberration test (OECD 473)
The in vitro chromosomal aberration (CA) test detects structural aberrations and
may give an indication for numerical chromosome aberrations (polyploidy)
Cultures of established cell lines or primary cell cultures. Cells in metaphase are
analysed for the presence of chromosomal aberrations
In vitro mammalian cell micronucleus test (OECD TG 487)
The assay detects micronuclei in the cytoplasm of interphase cells and typically
employs human or rodent cells lines or primary cell cultures.

Genotoxicity
Other in vitro assays
is anticipated that these reconstructed skin models could improve the predictive
value of a genotoxicity assessment compared with existing in vitro tests and,
therefore, could be
used as a follow-up test in case of positive results from the standard in vitro
genotoxicity testing
battery
3D RHE human skin model micronucleus and comet assay
Based on EpiDerm tissues, the tissues were topically exposed to test chemicals
for 3h followed by cell isolation and assessment of DNA damage using the comet
assay.
cover different kinds of genetic damage, namely the micronucleus test to detect
clastogenicity and aneugenicity, and the comet assay to detect DNA strand breaks,
incomplete repair sites and alkali labile sites

Carcinogenicity
Substances are defined as carcinogenic if after inhalation, ingestion,
dermal application or injection they induce (malignant) tumours, increase
their incidence or malignancy, or shorten the time of tumour occurrence. It
is generally accepted that carcinogenesis is a multihit/ multi-step process
from the transition of normal cells into cancer
Carcinogens have conventionally been divided into two categories
according to their presumed mode of action: genotoxic carcinogens and
non-genotoxic carcinogens (not involve direct alterations in DNA)
The two-year cancer bioassay in rodents is widely regarded as the goldstandard to evaluate cancer hazard and potency, although it is generally
known that this test has its limitations to predict human cancer risk.

Carcinogenicity
While in vitro and in vivo genotoxicity tests contribute to the assessement
of genotoxic carcinogens , there is a lack of tests available for the
assessment of non-genotoxic carcinogens.
The complexity of the carcinogenicity process makes it difficult to develop in
vitro alternative test models that mimic the full process, especially for nongenotoxic chemicals. The challenge in developing in vitro alternatives is
also heightened because of the complexity of the number of target organs.
It is expected that an integrated approach involving multiple in vitro models
will be needed, but a better understanding of the entire process is needed
before this will be possible

Carcinogenicity
In vitro cell Transformation assays (CTA assays)
n vitro CTAs have been shown to closely model some stages of the
multistage process of in vivo carcinogenesis. The phenomenon of
morphological transformation is characterised by changes in the behaviour
and growth of cultured cells allowing for progression to the next stage in the
transformation process, from a normal cell to a fully malignant cell.
A minimum of four phenotypic stages appears to be involved in cell
transformation
such tests are recommended only as supplements to core genotoxicity
tests for carcinogens. This is mainly because the molecular basis of the
transformed phenotype and its relationship to cancer in vivo is not fully
understood.

Toxicokinetics
-The TK phase begins with exposure and results in a certain
concentration of the ultimate toxicant at the target site (tissue dose).
This concentration is dependent on the absorption, distribution,
metabolism and excretion (ADME) of the substance (ECETOC, 2006).
ADME describes the uptake of a substance into the body and its
lifecycle within the body,

Toxicokinetics
-Even though TK is not a toxicological endpoint and is not specifically
required by REACH, the generation of TK information can be
encouraged as a means to interpret data, assist testing strategy and
study design, as well as category development, thus helping to
optimise test designs

-In assessing gained information in terms of human relevance, the

conservative approach of applying an assessment factor (default
approach) is used for taking into account uncertainties over
interspecies and intraspecies differences in sensitivity to a specific test
substance.

--route-to-route extrapolation is probably the most important use-case for

information on species- and route-specific ADME and, if available, systemic
exposure (whole-body TK)

-Anyway Animals are often poor models for humans due to sometimes wellknown qualitative and also quantitative differences in their physiology and
metabolism .

Toxicokinetics
-PBTK models are increasingly being used in the chemical risk assessment
process

- to take into account relevant in vivo differences (cross-species, cross-route

and inter-individual)

- to make better use of in vitro toxicity results.(In order to use human in vitro
toxicity data for human risk assessment, a stronger focus on internal
exposure (e.g. AUC and Cmax of the putative toxicant)
These models facilitate quantitative descriptions of the temporal
change in the concentration of chemical and/or its metabolites in
biological matrices (e.g., blood, tissue, urine, alveolar air) of the
exposed organism. PBTK models describe the organism as a set of
compartments that are characterized physiologically or empirically. Two
categories of parameters are needed in order to simulate the
toxicokinetics of a chemical in a PBTK model:
Physiological parameters that are chemical-independent, such as
cardiac output and organ blood flow (species-, sex- and age-specific

Toxicokinetics
-In vivo OECD 417 OECD TG 417 studies typically provide rather isolated
species-, dose-, and route-specific (mostly oral) data on absorption, tissue
distribution or metabolism. In rare cases, OECD TG 417 is used to give the
integrated TK profile of a substance, i.e. the concentration-time course of the
parent compound and its metabolites

-These studies are performed with radiolabelled material to allow for

comprehensive quantification of dose material and to aid potential
metabolite analysis.
The endpoints measured are

- Mass balance (summation of the percent of the administered

(radioactive) dose excreted in urine, faeces, and expired air, and the
percent present in tissues, residual carcass, and cage wash)

Toxicokinetics
-Only one ADME in vitro procedure has reached the level of an OECD
test guideline, e.g. dermal absorption in vitro (OECD TG 428, 2004). This
test method is been used as a stand-alone test method for several
regulatory requirements. However, to adequately measure the flux
across the dermal barrier for the purpose of integration into PBTK
modeling approaches, some additional work need to be carried out.

Toxicokinetics
Skin absorption OECD 428
vivo data may be used alone. It is advisable to contact the appropriate
regulatory authority to confirm
the most relevant and adequate test protocol(s) before conducting skin
absorption studies.

-Human skin from abdominal surgery (excised and cut into pieces of 2
cm x 2 cm)

-stratum corneum integrity will be measured for each dermatomed skin

sample by measuring the TEWL using evaporimeter

-The passive diffusion of chemicals (and therefore their dermal

absorption) is affected by temperature. The diffusion chamber and skin
samples will be maintained at a constant temperature

Toxicokinetics
The quantification of the substance is performed thanks to
The use of 14C or 3H molecule or a validated analytical method in in the
following compartments

-applicator (spreader, glass rod, loop, etc.)

donor chamber
dislodgeable dose from skin surface (washings, sponges, etc.).
stratum corneum, when sampled (tape strips, etc.)
skin preparation
receptor fluid and receptor chamber
Mass balance analysis and recovery data are to be provided. The
overall recovery of test substance (including metabolites) should be
within the range of 85-115%.

Toxicokinetics
Metabolic activity is not required to study dermal absorption of radiolabelled material, as this is a passive diffusion process. However, the
biotransformation of the compound in skin prior to
systemic absorption may be significant for some compounds. Where
maintenance of metabolic
activity is required (i.e., there is a requirement for the identification of
metabolites) it is necessary to
demonstrate that the relevant metabolic activity is maintained for the
duration of the study

Toxicokinetics
-Future:
-New Barrier models
-Gastrointestinal barrier absorption

Toxicokinetics
-Future:
-New Barrier models
- MucilAir (Epithelix SRL, Swiss)
MucilAir in vitro human 3D airway epithelium model was originally
designed for assessing the toxicity of nanoparticles and chemical
compounds
In a collaborative project EURL ECVAM optimised the test
system MucilAir to allow its use as an in vitro absorption barrier for
toxicokinetic purposes. The absorption of chemicals through the lung
barrier is analysed after apical or basolateral treatments

Toxicokinetics
-Future:
-Metabolism (biotransformation), which is one of the main difference
inter and intra-species, play a key role in the toxicokinetic and
toxicodynamic processes.
Proposed to use human cryopreserved HepaRG cell line and
cryopreserved human hepatocytes
The assay is based on cryopreserved human hepatocytes and
cryopreserved human metabolically competent cell line (HepaRG)
The endpoint induction of CYPs is measured following treatment
with test compounds and two reference compounds using a cocktail of
prototypical substrates for different CYP isoforms

Conclusions
- Importance of understanding Mechanisms
-Identify non-standard methods that can be associated with key events in one
or more AOPs.

-No stand alone test

-A structured approach which integrates and weights all relevant
existing data and inform about additional data needs to enable
(regulatory) decisions

-Toxicokinetics
-No toxicological test is perfect including the animal tests it is
important to know their strengths and limitations

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