Advancing Safety Science and

152-154 Curtain Road, Shoreditch (EC2A 3AT

Health Research with Human BiologyBased Tools of the 21st Century
Troy Seidle
Director, Research & Toxicology Department
tseidle@hsi.org
Humane Society International
hsi.org/endanimaltesting

About HSI
Expert team: toxicology, ecotoxicology,
pharmacology, biochemistry, neuroscience,
endocrinology, law, regulatory science, etc.
Present in Korea, China, Japan, India,
Australia, Europe, United States, Canada,
Mexico, Brazil, Latin America, and beyond
Working with research institutes,
companies, government regulators and
other stakeholders

HSI is the leading

international NGO
working to advance
non-animal safety
testing and bioscience
research worldwide.

HSI regulatory science team representation

OECD Test Guidelines, Chemicals & AOP development programs

EU Competent Authorities for REACH and Classification and Labeling

European Chemicals Agency Member State Committee, Endocrine Disruptors
Expert Group, etc.
USEPA Pesticide Program Workgroup on 21st Century Toxicology
USTR Trade and Environment Policy Committee & TTIP negotiations
2015 Public Forum for Science Technology Assessment by Ministry of Science,
ICT and Future Planning and Korea Institute of S&T Evaluation and Planning
Chinese Environmental Mutagen Society 21st Century Toxicology Group
NTP Scientific Advisory Committee on Alternative Toxicological Methods
European Union Reference Laboratory for Alternatives (EURL-ECVAM)

International Cooperation on Cosmetics Regulation & national laws

International Conference on Harmonization (via ICAPPP)
Human Toxicology Project Consortium
and others

HSI research & toxicology portfolio worldwide

HSIs vision
Near-term reduction in animal use through
uptake of 3R best practices in product
sector regulations (cosmetics, chemicals,
pesticides, etc.)

Shift to a fully human

biology-based paradigm
based on understanding
of adverse outcome
pathways (AOPs)

HSI Korea research & toxicology portfolio

Ending cosmetics animal testing and trade

Acceptance of available alternatives and

accelerating transition to 21st century paradigm
Exporting 21st century paradigm from
toxicology to wider bioscience research sector

1. Ending cosmetics animal testing & trade

APRIL 2012

HSI and KARA launch effort to align Korean cosmetics policy

with EU precedent (as worlds largest beauty market)
MARCH 2015

Congresswoman Moon Jeong-lim introduces bill that would:

Legally require use of available animal testing
alternatives for cosmetics
Prohibit sale of cosmetics and ingredients
subject to new animal testing (with exceptions)
Call on regulators to work toward having more
alternative methods available

2a. Evolving approaches to testing and assessment

Significant progress in development and
validation of 3R testing tools (cell and
computer models), as well as non-testing
approaches (waivers, read-across)
Deployed according to integrated testing
strategies using weight-of-evidence
Ongoing paradigm shift based on AOP
knowledge
Regular updates to regulatory data
requirements needed across product
sectors (cosmetics, chemicals, pesticides)
to keep pace with scientific progress

OECD guideline tests with replacement potential

Endpoint

Test methods

OECD guideline (year)

Skin corrosion /
irritation

Reconstructed Human Epithelium

(RHE), CORROSITEX

431 (2004), 435 (2006), 439

(2013)

Eye corrosion /
irritation

BCOP, ICE, IRE, Fluorescein

Leakage, EpiOcular

437 & 438 (2013), 460

(2012), 491, 492 (2015)

Skin sensitization

DPRA, KeratinoSens (ARE-nrf2

luciferase), h-CLAT

442C & 442D (2015), draft

442E

Skin absorption

Human post-surgical skin; RHE

models

428 (2004)

Phototoxicity

3T3 Neutral Red Uptake (NRU)

432 (2004)

Mutagenicity

Ames, Chromosomal Aberration,

Micronucleus, etc.

471, 473, 476 & 479 (1986),

487 (2010), 490 (2015)

Acute fish toxicity

Fish embryo test

236 (2013)

Acceptance of available alternatives in regulation

HSI has led successful negotiations to revise chemical, pesticide and other
regulations in the EU, US and elsewhere, achieving dramatic reductions in
obsolete animal test requirements
Opportunity to revise K-REACH data requirements to ensure:
Uptake of all applicable OECD 3R guideline methods, as well as other
scientifically supported alternative testing (or non-testing) strategies
Deletion of needlessly redundant in vivo studies (e.g., multiple exposure routes
and/or species)
Endpoint-combining (e.g., in vivo micronucleus as part of a 28-day study)
Adopting more efficient study designs (e.g., extended 1-gen repro study)

Ongoing 3R revisions to EU REACH data requirements

HSI suggestions for updating K-REACH requirements

Endpoint

3R best practice

Reproductive
toxicity

Extended 1-generation study (EOGRTS) instead

of 2-gen (- 1,200 animals = 46% reduction)

Genetic toxicity,
micronucleus

Carcinogenicity

Assess in combination with a repeated dose

toxicity study rather than 2 separate studies
(- 80 animals)
Waive study requirement if substance is
unclassified (non-toxic) via oral route
(- 30 animals)
Waive mouse study (- 400 animals)

Developmental
toxicity

Waive rat studyif no concerns from rabbit

developmental or rat EOGRTS (- 1,300 animals)

Acute toxicity,
fish

Tiered threshold strategy (70% reduction)

etc.

Ongoing discussion of other available

alternatives

Acute toxicity,
dermal

EU

KR

2b. Advancing 21st century toxicology

Founded and co-led the AXLR8 (accelerate) project in
developing a strategic European research agenda to
advance animal-free safety testing
Participated in political negotiations for the EUs 201420 research funding framework
More than 250 million in new funding made available
for human-relevant science and infrastructures

Currently working to secure similar public and private

investments in other science- and innovation-based
economies (USA, Korea, Japan, India, Canada, etc.)

Advancing 21st century toxicology in Korea

Promoting prioritized funding for development and
application of AOP-based in vitro, in silico, and
other human biology-based tools in Korea
14.18 billion budget proposed by Cong. Moon

Supporting conferences on 21st century,

non-animal toxicology and health research
2016 Pan-Asian Alternatives Congress
15-18 November 2016, Fukuoka, Japan
Korea represented on program committee

Encouraging increased Korean involvement

in the OECD global research effort to map
human AOPs

3. Toward a 21st century medical research paradigm

Funding scholarly reviews in several human
disease areas (asthma, Alzheimers, autism,
autoimmune disease, etc.), identifying failing
animal models and proposing scientific
roadmap for each disease area based on
AOP knowledge and human biology-based
methods
Beginning a dialogue with research funding
bodies aimed at shifting resources toward
more human-relevant lines of investigation
Secured substantial revisions to the EUs
animal experiments directive and working
toward similar revisions in Korea, Japan,
Brazil and elsewhere

Toward a 21st century medical research paradigm

Rallying global thought leaders to develop new strategic science agenda
Call for a funding shift toward a human
pathway-based paradigm, co-authored by
HSI research & toxicology dept. scientists
and colleagues representing

Opportunities for collaboration

General Assembly Members
Prioritize substantial, long-term funding for human biology-based health
research, toxicological testing tools and infrastructures

Ministry of Environment & National Institute of Environmental Research

Revision of K-REACH data requirements and test guidelines

Ministry of Agriculture, Food and Rural Affairs

Revision of pesticide data requirements & 3Rs strategy under 5-year plan

Ministry of Food and Drug Safety

3Rs strategy & ongoing reclassification of functional/ordinary cosmetics

Korea Centre for the Validation of Alternative Methods

Expand inter-agency representation & financial investment in KoCVAM

Industry, research institutes & academic societies

Increased involvement in Korean & OECD AOP programs

Looking forward to future collaboration

Troy Seidle
Director
Research & Toxicology Department
tseidle@hsi.org
Borami Seo
Policy Advisor, Research & Toxicology Department
Seoul, Korea
bseo@hsi.org

---------------------------------------------

---------------------------------------------

In 2012, Humane Society International (HSI) submitted detailed technical

recommendations to the European Commission in support of revisions to
registration data requirements under its REACH chemicals regulation to spare
millions of animals while providing the same level of regulatory scrutiny of
chemicals. In response, the EU has revised REACH to include a reduction
alternative for reproductive toxicity, and is in the process of implementing animal
replacement alternatives for skin and eye irritation/corrosion, skin sensitization,
as well as a waiver strategy for dermal acute toxicity. HSI urges Korean authorities
to implement these and other available 3R alternatives under K-REACH.

2012 (HSI) Eurpean Commission REACH

.

. reproductive toxicity
, skin & eye irritation/corrosion, skin
sensitization dermal
acute toxicity waiver strategy . HSI (
) 3Rs .

21.2.2015

EN

Official Journal of the European Union

L 50/1

II
(Non-legislative acts)

REGULATIONS
COMMISSION REGULATION (EU) 2015/282
of 20 February 2015
amending Annexes VIII, IX and X to Regulation (EC) No 1907/2006 of the European Parliament
and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals
(REACH) as regards the Extended One-Generation Reproductive Toxicity Study
(Text with EEA relevance)
THE EUROPEAN COMMISSION,

Having regard to the Treaty on the Functioning of the European Union,

Having regard to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006
concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European
Chemicals Agency, amending Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and
Commission Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission Directives
91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (1), and in particular Article 13(2) thereof,
Whereas:
(1)

Article 13(2) of Regulation (EC) No 1907/2006 provides that testing methods used to generate information on
intrinsic properties of substances required by that Regulation are to be regularly reviewed and improved with a
view to reducing testing on vertebrate animals and the number of animals involved. The principles of
replacement, reduction and refinement, enshrined in Directive 2010/63/EU of the European Parliament and of
the Council (2) should be taken into account in the design of the test methods, in particular when appropriate
validated methods become available to replace, reduce or refine animal testing. Following that review, Council
Regulation (EC) No 440/2008 (3) and the Annexes to Regulation (EC) No 1907/2006 are to be amended, if
relevant, so as to replace, reduce or refine animal testing.

(2)

Pursuant to Regulation (EC) No 1907/2006, a two-generation reproductive toxicity study is to be used

investigate the reproductive toxicity of chemical substances to fulfil the standard information requirements
point 8.7.3 of Annexes IX and X to that Regulation. Furthermore, column 2 of point 8.7.1 of Annex VIII
Regulation (EC) No 1907/2006 provides that the two-generation reproductive toxicity study is a possibility
assess the cases where there are serious concerns about the potential for adverse effects on fertility
development.

(3)

The Extended One-Generation Reproductive Toxicity Study (4) (EOGRTS) is a new test method developed to
assess the reproductive toxicity of chemical substances. This test method was adopted by the Organisation for
Economic Cooperation and Development (OECD) in July 2011. EOGRTS is a modular test method, where
breeding and assessment of a second filial (F2) generation and testing for developmental neurotoxicity (DNT) and
developmental immunotoxicity (DIT) constitute distinct and independent modules.

to
in
to
to
or

(1) OJ L 396, 30.12.2006, p. 1.

(2) Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for
scientific purposes (OJ L 276, 20.10.2010, p. 33).
(3) Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the
European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 142,
31.5.2008, p. 1).
(4) OECD Test Guideline 443.

L 50/2

EN

Official Journal of the European Union

21.2.2015

(4)

EOGRTS is considered to offer a number of advantages in comparison to the two-generation reproductive

toxicity study. It assesses a greater number of animals of the first filial (F1) generation and addresses additional
parameters, thus improving the sensitivity and level of information that can be obtained from the test.
Furthermore, as breeding of the F2 generation is not part of the basic test design, a significant reduction in the
number of animals used is achieved if this design is used.

(5)

EOGRTS was included in Regulation (EC) No 440/2008 by Commission Regulation (EU) No 900/2014 (1).
Annexes IX and X to Regulation (EC) No 1907/2006 should be amended to specify how the new test method is
to be used for the purposes of Regulation (EC) No 1907/2006. To this end, a sub-group of the Commission
Expert Group consisting of Competent Authorities for the REACH and the classification and labelling of chemical
substances Regulations (the Expert Group) was created in 2011. Based on the scientific recommendations of this
Expert Group, the EOGRTS should become the preferred test method to address the standard information
requirement defined in column 1 of point 8.7.3 of Annexes IX and X to Regulation (EC) No 1907/2006 instead
of the two-generation reproductive toxicity study (B.35).

(6)

The standard information requirement in Annexes IX and X to Regulation (EC) No 1907/2006 should be limited
to the basic configuration of EOGRTS. Nevertheless, in certain specific cases, where justified, the registrant should
be able to propose and the European Chemicals Agency (ECHA) should be able to request the performance of the
F2 generation, as well as the DNT and DIT cohorts.

(7)

It should be ensured that the reproductive toxicity study carried-out under point 8.7.3 of Annexes IX and X to
Regulation (EC) No 1907/2006 will allow adequate assessment of possible effects on fertility. The premating
exposure duration and dose selection should be appropriate to meet risk assessment and classification and
labelling purposes as required by Regulation (EC) No 1907/2006 and Regulation (EC) No 1272/2008 of the
European Parliament and of the Council (2).

(8)

Considering that the remaining scientific concerns as regards the value of the F2 generation should be clarified
on the basis of empirical data, and that substances potentially presenting the highest risk to consumers and
professional users should be assessed on the basis of a conservative approach, the production and assessment of
the F2 generation should be triggered for certain substances on a case-by-case basis. The Expert Group
recommended that an exposure based trigger, associated with uses leading to exposures of consumers and
professional users should be implemented in the relevant points of Annexes IX and X to Regulation (EC)
No 1907/2006. Additional criteria, based on evidence indicating that a substance is of concern as a function of
the available toxicity and toxicokinetic information, should be included to further optimise the selection of
substances for which the F2 generation should be produced and subjected to testing.

(9)

Developmental Neurotoxicity and developmental immunotoxicity are regarded as important and relevant develop
mental toxicity endpoints, which could be further investigated. However, analysing the DNT and DIT cohorts
entails significant additional cost as well as technical and practical difficulties for testing laboratories. Therefore, it
is considered appropriate to subject the analysis of the DIT and DNT cohorts, or only one of them, to specific
concern-driven scientific triggers. Specific rules for the adaptation of the information requirement defined in
point 8.7.3 of Annexes IX and X to Regulation (EC) No 1907/2006 should be introduced, so as to trigger the
immunotoxicity and neurotoxicity testing. In cases where the available information on a substance indicates a
particular concern on neurotoxicity or immunotoxicity, the inclusion of the DNT and the DIT cohorts, or only
one of them, justified on a case-by-case basis, should be possible. Evidence supporting these concerns could
originate from existing information derived from in vivo or non-animal approaches, from the knowledge of
relevant mechanisms/modes of action of the substance itself, or from existing information on structurally related
substances. Therefore, if any such particular concerns are justified, the registrant should be required to propose,
and ECHA should be able to request the performance of the DNT and DIT cohorts, or only one of them.

(1) Commission Regulation (EU) No 900/2014 of 15 July 2014 amending, for the purpose of its adaptation to technical progress,
Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of
the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 247, 21.8.2014, p. 1).
2
( ) Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and
packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC)
No 1907/2006 (OJ L 353, 31.12.2008, p. 1).

21.2.2015

EN

Official Journal of the European Union

L 50/3

(10)

Point 8.7.3 of Annex IX to Regulation (EC) No 1907/2006 requires performing a reproductive toxicity study,
only if there are concerns arising from adverse effects previously detected on reproductive organs or tissues. That
point provides that only 28- and 90-day repeated dose toxicity studies can be the source of such information.
Given that also reproductive toxicity screening studies such as OECD Test Guideline 421 or Test Guideline 422,
or other studies with repeated dose administration can provide indications on adverse effects on relevant
reproductive parameters, which may justify the need to follow-up by performing an EOGRTS, column 1 of
point 8.7.3 should be modified to allow such additional studies to be considered.

(11)

In order to avoid imposing a disproportionate burden on the economic operators who may have already
performed the tests or acquired results of two-generation reproductive toxicity study, as well as for animal welfare
reasons, the robust study summaries of those studies that were initiated before the date of the entry into force of
this Regulation should be considered appropriate to address the standard information requirement in point 8.7.3
of Annexes IX and X to Regulation (EC) No 1907/2006.

(12)

For reasons of consistency, point 8.7.1, column 2 of Annex VIII to Regulation (EC) No 1907/2006 should be
amended in order to change the cross-reference to the study required under point 8.7.3 of Annex IX to
Regulation (EC) No 1907/2006 from the two-generation reproductive toxicity study to EOGRTS.

(13)

ECHA, in close cooperation with Member States and stakeholders, should further develop guidance documents
for the application of EOGRTS for the purposes of Regulation (EC) No 1907/2006, including on the application
of the criteria for F2 and DNT/DIT cohorts. In doing so, ECHA should take full account of the work carried out
in OECD, as well as in other relevant scientific and expert groups. Furthermore, when determining deadlines by
which dossier updates providing results of EOGRTS are to be submitted, ECHA should take due account of the
market availability of this testing service.

(14)

Regulation (EC) No 1907/2006 should therefore be amended accordingly.

(15)

The measures provided for in this Regulation are in accordance with the opinion of the Committee established
under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:

Article 1
Annexes VIII, IX and X to Regulation (EC) No 1907/2006 are amended in accordance with the Annex to this
Regulation.
Article 2
This Regulation shall enter into force on the twentieth day following that of its publication in the Official Journal of the
European Union.

This Regulation shall be binding in its entirety and directly applicable in all Member States.
Done at Brussels, 20 February 2015.
For the Commission
The President
Jean-Claude JUNCKER

L 50/4

EN

Official Journal of the European Union

21.2.2015

ANNEX

Regulation (EC) No 1907/2006 is amended as follows:

(1) in Annex VIII, in the table setting out the toxicological information, in column 2 (Specific Rules for Adaptation from
column 1), point 8.7.1 is replaced by the following:
8.7.1. This study does not need to be conducted if:
the substance is known to be a genotoxic carcinogen and appropriate
risk management measures are implemented, or
the substance is known to be a germ cell mutagen and appropriate
risk management measures are implemented, or
relevant human exposure can be excluded in accordance with Annex XI
section 3, or
a pre-natal developmental toxicity study (Annex IX, 8.7.2) or, either an
Extended One-Generation Reproductive Toxicity Study (B.56, OECD
TG 443) (Annex IX, section 8.7.3) or a two-generation study (B.35,
OECD TG 416), is available.
If a substance is known to have an adverse effect on fertility, meeting the
criteria for classification as toxic for reproduction category 1A or 1B: May
damage fertility (H360F), and the available data are adequate to support a
robust risk assessment, then no further testing for fertility will be neces
sary. However, testing for developmental toxicity must be considered.
If a substance is known to cause developmental toxicity, meeting the cri
teria for classification as toxic for reproduction category 1A or 1B: May
damage the unborn child (H360D), and the available data are adequate to
support a robust risk assessment, then no further testing for developmen
tal toxicity will be necessary. However, testing for effects on fertility must
be considered.
In cases where there are serious concerns about the potential for adverse
effects on fertility or development, either an Extended One-Generation Re
productive Toxicity Study (Annex IX, section 8.7.3) or a pre-natal develop
mental toxicity study (Annex IX, section 8.7.2) may, as appropriate, be
proposed by the registrant instead of the screening study.

(2) in Annex IX, in the table setting out the toxicological information, in column 1 (Standard Information Requirement)
and column 2 (Specific Rules for Adaptation from column 1) point 8.7.3 is replaced by the following:
8.7.3. Extended One-Generation Reproduc 8.7.3. An Extended One-Generation Reproductive Toxicity Study
tive Toxicity Study (B.56 of the Com
with the extension of cohort 1B to include the F2 generation
mission Regulation on test methods
shall be proposed by the registrant or may be required by the
as specified in Article 13(3) or OECD
Agency in accordance with Article 40 or 41, if:
443), basic test design (cohorts 1A
(a) the substance has uses leading to significant exposure of
and 1B without extension to include a
consumers or professionals, taking into account, inter alia,
F2 generation), one species, most ap
consumer exposure from articles, and
propriate route of administration,
(b) any of the following conditions are met:
having regard to the likely route of
human exposure, if the available re
the substance displays genotoxic effects in somatic cell
peated dose toxicity studies (e.g.
mutagenicity tests in vivo which could lead to classify
28-day or 90-day studies, OECD 421
ing it as Mutagen Category 2, or
or 422 screening studies) indicate ad
there are indications that the internal dose for the sub
verse effects on reproductive organs
stance and/or any of its metabolites will reach a steady
or tissues or reveal other concerns in
state in the test animals only after an extended expo
relation with reproductive toxicity.
sure, or
there are indications of one or more relevant modes of
action related to endocrine disruption from available
in vivo studies or non-animal approaches.

21.2.2015

EN

Official Journal of the European Union

L 50/5

An Extended One-Generation Reproductive Toxicity Study in

cluding cohorts 2A/2B (developmental neurotoxicity) and/or
cohort 3 (developmental immunotoxicity) shall be proposed
by the registrant or may be required by the Agency in accord
ance with Article 40 or 41, in case of particular concerns on
(developmental) neurotoxicity or (developmental) immuno
toxicity justified by any of the following:
existing information on the substance itself derived from
relevant available in vivo or non-animal approaches (e.g.
abnormalities of the CNS, evidence of adverse effects on
the nervous or immune system in studies on adult animals
or animals exposed prenatally), or
specific mechanisms/modes of action of the substance
with an association to (developmental) neurotoxicity
and/or (developmental) immunotoxicity (e.g. cholinesterase
inhibition or relevant changes in thyroidal hormone levels
associated to adverse effects), or
existing information on effects caused by substances struc
turally analogous to the substance being studied, suggest
ing such effects or mechanisms/modes of action.
Other studies on developmental neurotoxicity and/or develop
mental immunotoxicity instead of cohorts 2A/2B (develop
mental neurotoxicity) and/or cohort 3 (developmental immu
notoxicity) of the Extended One-Generation Reproductive
Toxicity Study may be proposed by the registrant in order to
clarify the concern on developmental toxicity.
Two-generation reproductive toxicity studies (B.35, OECD
TG 416) that were initiated before 13 March 2015 shall be
considered appropriate to address this standard information
requirement.
The study shall be performed on one species. The need to per
form a study at this tonnage level or the next on a second
strain or a second species may be considered and a decision
should be based on the outcome of the first test and all other
relevant available data.

(3) in Annex X, in the table setting out the toxicological information, in column 1 (Standard Information Requirement)
and column 2 (Specific Rules for Adaptation from column 1) point 8.7.3 is replaced by the following:

8.7.3. Extended One-Generation Reproduc 8.7.3. An Extended One-Generation Reproductive Toxicity Study
tive Toxicity Study (B.56 of the Com
with the extension of cohort 1B to include the F2 generation
mission Regulation on test methods
shall be proposed by the registrant or may be required by the
as specified in Article 13(3) or OECD
Agency in accordance with Article 40 or 41, if:
443), basic test design (cohorts 1A
(a) the substance has uses leading to significant exposure of
and 1B without extension to include a
consumers or professionals, taking into account, inter alia,
F2 generation), one species, most ap
consumer exposure from articles, and
propriate route of administration,
(b) any of the following conditions are met:
having regard to the likely route of
human exposure, unless already pro
the substance displays genotoxic effects in somatic cell
vided as part of Annex IX require
mutagenicity tests in vivo which could lead to classify
ments.
ing it as Mutagen Category 2, or
there are indications that the internal dose for the sub
stance and/or any of its metabolites will reach a steady
state in the test animals only after an extended expo
sure, or
there are indications of one or more relevant modes of
action related to endocrine disruption from available
in vivo studies or non-animal approaches.

L 50/6

EN

Official Journal of the European Union

21.2.2015

An Extended One-Generation Reproductive Toxicity Study in

cluding cohorts 2A/2B (developmental neurotoxicity) and/or
cohort 3 (developmental immunotoxicity) shall be proposed
by the registrant or may be required by the Agency in accord
ance with Article 40 or 41, in case of particular concerns on
(developmental) neurotoxicity or (developmental) immuno
toxicity justified by any of the following:
existing information on the substance itself derived from
relevant available in vivo or non-animal approaches (e.g.
abnormalities of the CNS, evidence of adverse effects on
the nervous or immune system in studies on adult animals
or animals exposed prenatally), or
specific mechanisms/modes of action of the substance
with an association to (developmental) neurotoxicity
and/or (developmental) immunotoxicity (e.g. cholinesterase
inhibition or relevant changes in thyroidal hormone levels
associated to adverse effects), or
existing information on effects caused by substances struc
turally analogous to the substance being studied, suggest
ing such effects or mechanisms/modes of action.
Other studies on developmental neurotoxicity and/or develop
mental immunotoxicity instead of cohorts 2A/2B (develop
mental neurotoxicity) and/or cohort 3 (developmental immu
notoxicity) of the Extended One-Generation Reproductive
Toxicity Study may be proposed by the registrant in order to
clarify the concern on developmental toxicity.
Two-generation reproductive toxicity studies (B.35, OECD
TG 416) that were initiated before 13 March 2015 shall be
considered appropriate to address this standard information
requirement.

EUROPEAN
COMMISSION

Brussels, XXX
[](2015) XXX draft

COMMISSION REGULATION (EU) /

of XXX
amending Annexes VII and VIII to Regulation (EC) No 1907/2006 of the European
Parliament and of the Council on the Registration, Evaluation, Authorisation and
Restriction of Chemicals (REACH) as regards skin corrosion/irritation, serious eye
damage/eye irritation, skin sensitisation and acute toxicity

(Text with EEA relevance)

EN

EN

COMMISSION REGULATION (EU) /

of XXX
amending Annexes VII and VIII to Regulation (EC) No 1907/2006 of the European
Parliament and of the Council on the Registration, Evaluation, Authorisation and
Restriction of Chemicals (REACH) as regards skin corrosion/irritation, serious eye
damage/eye irritation, skin sensitisation and acute toxicity

(Text with EEA relevance)

THE EUROPEAN COMMISSION,

Having regard to the Treaty on the Functioning of the European Union,
Having regard to Regulation (EC) No 1907/2006 of the European Parliament and of the
Council of 18 December 2006 concerning the Registration, Evaluation, Authorisation and
Restriction of Chemicals (REACH), establishing a European Chemicals Agency, amending
Directive 1999/45/EC and repealing Council Regulation (EEC) No 793/93 and Commission
Regulation (EC) No 1488/94 as well as Council Directive 76/769/EEC and Commission
Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC (1), and in particular Articles
13(2) and 131 thereof,
Whereas:
(1)

Article 13(2) of Regulation (EC) No 1907/2006 provides that test methods used to
generate information on intrinsic properties of substances required by that Regulation
are to be regularly reviewed and improved with a view to reducing testing on
vertebrate animals and the number of animals involved. When appropriate validated
test methods become available, the Commission Regulation (EC) No 440/20082 and
the Annexes to Regulation (EC) No 1907/2006 should be amended, if relevant, so as
to replace, reduce or refine animal testing. The principles of replacement, reduction
and refinement, enshrined in Directive 2010/63/EU of the European Parliament and of
the Council3 should be taken into account.

(2)

Regulation (EC) No 1907/2006 establishes requirements for the registration of

substances manufactured or imported in the Union on their own, in mixtures or
articles. The registrants have to provide the information required by Regulation (EC)
No 1907/2006, as appropriate, in order to fulfil the registration requirements.

(3)

Pursuant to Regulation (EC) No 1907/2006, in vivo studies are required for the
generation of information on skin sensitisation in point 8.3 of Annex VII to Regulation
(EC) No 1907/2006 and for skin irritation and eye irritation in points 8.1 and 8.2 of
Annex VIII to Regulation (EC) No 1907/2006.

OJ L 396, 30.12.2006, p. 1.
Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to
Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH) (OJ L 142, 31.5.2008, p. 1).
Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the
protection of animals used for scientific purposes (OJ L 276, 20.10.2010, p. 33).

EN

EN

EN

(4)

In recent years, significant scientific progress has been made in the development of
alternative test methods for skin corrosion/ irritation, serious eye damage/eye irritation
and skin sensitisation. A number of test guidelines for alternative test methods have
been internationally agreed by the Organisation for Economic Co-operation and
Development (OECD), and have been, or are foreseen to be, included in Commission
Regulation (EC) No 440/2008.

(5)

For skin corrosion/skin irritation, adequate information for the classification and/or
risk assessment of a substance may be obtained in most cases solely on the basis of in
vitro studies. A conclusion may be drawn on the basis of one test, if the result allows
immediate classification, or from a combination of two tests, one for skin irritation and
one for skin corrosion. In vivo studies may still be required in some exceptional cases,
e.g. when the substance tested falls outside the applicability domain of the test
methods or when no conclusive results can be obtained from a comprehensive set of in
vitro tests.

(6)

For serious eye damage/eye irritation, a set of in vitro test methods exists which would
be sufficient in many cases to obtain information adequate for classification and/or
risk assessment of substances. A conclusion about the potential of a substance to cause
such eye effects may be drawn on the basis of one test, if the result allows immediate
classification, or from a combination of two or more tests. In vivo studies may still be
required in some cases, e.g. when the substance tested falls outside the applicability
domain of the test methods or when no conclusive results can be obtained from a
comprehensive set of in vitro tests.

(7)

Points 8.1 and 8.2 of Annex VIII should thus be amended in order to remove the
standard information requirement for an in vivo study for skin irritation/corrosion and
serious eye damage/eye irritation.

(8)

For skin sensitisation, several alternative test methods have been validated by the
European Union Reference Laboratory for Alternatives to Animal Testing (EURL
ECVAM) and/or internationally agreed by the Organisation for Economic Cooperation and Development (OECD). These test methods may allow the generation of
adequate information to assess whether a substance causes skin sensitisation without
the need to resort to in vivo testing, when applied in an appropriate combination in the
framework of an integrated approach to testing and assessment (IATA). To reduce
animal testing, point 8.3 of Annex VII to Regulation (EC) No 1907/2006 should
explicitly allow waiving the in vivo test for skin sensitisation, if adequate information
may be obtained through non-animal test methods.

(9)

In addition, the standard information requirements and adaptation rules in points 8.1,
8.2 and 8.3 of Annex VII, and the adaptation rules in points 8.1 and 8.2 of Annex
VIII should be revised in order to remove redundancies with rules set by Annex VI
and Annex XI and in the introductory parts of Annexes VII and VIII as regards the
review of available data, the waiving of studies for a toxicological endpoint if the
available information indicates that the substance meets the criteria for classification
for that toxicological endpoint, or to clarify the intended meaning as regards the
waiving of studies for substances that are flammable under certain conditions. Where
reference is made to the classification of substances, adaptation rules should be
updated to reflect the terminology used in Regulation (EC) No 1272/20084.

Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on
classification, labelling and packaging of substances and mixtures, amending and repealing Directives

EN

(10)

Point 8.5 of Annex VIII to Regulation (EC) No 1907/2006 provides a standard

information requirement for substances other than gases on acute toxicity by the oral
route and, depending on the likely route of human exposure, by at least one additional
route (inhalation or dermal). Recent scientific analysis of available data from in vivo
acute toxicity studies have shown that substances that are not toxic via the oral route
may be expected with high certainty to be also non-toxic via the dermal route.
Therefore, testing those substances via the dermal route does not provide essential
information for their safety assessment. Point 8.5 of Annex VIII to Regulation (EC)
No 1907/2006 should thus be amended to provide for the possibility to waive the
dermal test for such substances.

(11)

ECHA, in cooperation with Member States and stakeholders, should further develop
guidance documents for the application of the test methods and waiving possibilities
for the standard information requirements provided by this Regulation for the purposes
of Regulation (EC) No 1907/2006. In doing so, ECHA should take full account of the
work carried out in OECD, as well as in other relevant scientific and expert groups.

(12)

Regulation (EC) No 1907/2006 should therefore be amended accordingly.

(13)

The measures provided for in this Regulation are in accordance with the opinion of the
Committee established under Article 133 of Regulation (EC) No 1907/2006,

HAS ADOPTED THIS REGULATION:

Article 1
Annexes VII and VIII to Regulation (EC) No 1907/2006 are amended in accordance with the
Annex to this Regulation.
Article 2
This Regulation shall enter into force on the [] day following that of its publication in the
Official Journal of the European Union.
This Regulation shall be binding in its entirety and directly applicable in all Member States.
Done at Brussels,

For the Commission

The President
[]

67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 (OJ L 353, 31.12.2008, p.
1)

EN

EN

EN
ANNEX
Point 8.1. of Annex VII shall be replaced by the following:
"8.1. Skin corrosion/
irritation

8.1. The study/ies do(es) not need to be conducted if:

the substance is a strong acid (pH 2,0) or base (pH
11,5) and it is classified as corrosive to skin (Cat. 1), or
the substance is spontaneously flammable in air or in
contact with water or moisture at room temperature, or
the substance is classified as acutely toxic by dermal route
(Cat. 1) , or
an acute toxicity study by the dermal route does not
indicate skin irritation up to the limit dose level (2 000 mg/kg
body weight).
If results from one of the studies under points 8.1.1 or 8.1.2
already allow a conclusive decision on the classification of a
substance, or on the absence of skin irritation potential, the
second study need not be conducted.

8.1.1 Skin corrosion, in vitro

8.1.2 Skin irritation, in vitro "

Point 8.2. of Annex VII shall be replaced by the following:

"8.2. Serious eye damage/eye
irritation

8.2.1 Serious eye damage/

eye irritation, in vitro"

8.2. These study/ies do(es) not need to be conducted if:

- the substance is classified as corrosive to skin and the
registrant classifies it as causing serious eye damage (Cat. 1),
or
the substance is a strong acid (pH 2,0) or base (pH
11,5) and it is classified as causing serious eye damage (Cat.
1), or
the substance is spontaneously flammable in air or in
contact with water or moisture at room temperature.

If results from a first in vitro study do not allow a conclusive

decision on the classification of a substance, or on the
absence of eye irritation potential, other in vitro studies for
this endpoint shall be considered.

Point 8.3. of Annex VII shall be replaced by the following:

"8.3. Skin sensitisation

This study does not need to be conducted if:

the substance is classified for skin corrosion, or
the substance is a strong acid (pH 2,0) or base (pH
11,5), or
the substance is spontaneously flammable in air or in
contact with water or moisture at room temperature.

8.3.1 Skin sensitisation, in

vivo.

This study does not need to be conducted if:

- sufficient information that is adequate for classification
and/or risk assessment is available from non-animal
approaches.
The Murine Local Lymph Node Assay (LLNA) is the firstchoice method for in vivo testing. Only in exceptional
circumstances should another test be used. Justification for
the use of another test shall be provided."

Point 8.1. of Annex VIII shall be replaced by the following:

"8.1. Skin corrosion/
irritation

8.1. An in vivo study for skin corrosion/irritation shall be

considered only if the in vitro studies under point 8.1.1. and
8.1.2. in Annex VII are not applicable, or the results of these
studies are not adequate for classification and/or risk
assessment.
The study does not need to be conducted if:
the substance is a strong acid (pH 2,0) or base (pH
11,5) and it is classified as corrosive to skin (Cat. 1), or
the substance is spontaneously flammable in air or in
contact with water or moisture at room temperature, or
the substance is classified as acutely toxic by dermal route
(Cat. 1), or
an acute toxicity study by the dermal route does not
indicate skin irritation up to the limit dose level (2 000 mg/kg
body weight)."

Point 8.2. of Annex VIII shall be replaced by the following:

"8.2. Serious eye
damage/eye irritation

8.2. An in vivo study for eye corrosion/irritation shall be

considered only if the in vitro studies under point 8.2.1. in
Annex VII are not applicable, or the results obtained from
these studies are not adequate for classification and/or risk
assessment.
The study does not need to be conducted if:
the substance is classified as corrosive to the skin,
resulting in a classification as causing serious eye damage
(Cat. 1), or
the substance is a strong acid (pH 2,0) or base (pH
11,5) and classified as causing serious eye damage (Cat. 1),
or
the substance is spontaneously flammable in air or in
contact with water or moisture at room temperature."

Point 8.5. of Annex VIII shall be replaced by the following:

"8.5. Acute toxicity

8.5. The study/ies do(es) not generally need to be conducted if:

the substance is classified as corrosive to the skin.
In addition to the oral route (Annex VII, 8.5.1), for substances
3

other than gases, the information mentioned under 8.5.2 to 8.5.3

shall be provided for at least one other route. The choice for the
second route will depend on the nature of the substance and the
likely route of human exposure. If there is only one route of
exposure, information for only that route need be provided.
8.5.2. By inhalation

8.5.2. Testing by the inhalation route is appropriate if exposure of

humans via inhalation is likely taking into account the vapour
pressure of the substance and/or the possibility of exposure to
aerosols, particles or droplets of an inhalable size.

8.5.3. By dermal route

8.5.3. Testing by the dermal route is appropriate if:

(1) inhalation of the substance is unlikely; and
(2) skin contact in production and/or use is likely; and
(3) the physicochemical and toxicological properties suggest
potential for a significant rate of absorption through the skin.
Testing by the dermal route does not need to be conducted if:
- the substance does not meet the criteria for classification as
acutely toxic or STOT SE by the oral route and
- no systemic effects have been observed in in vivo studies with
dermal exposure (e.g. skin irritation, skin sensitisation) or, in the
absence of an in vivo study by the oral route, no systemic effects
after dermal exposure are predicted on the basis of non-testing
approaches (e.g. read across, QSAR studies). "

2015.10.28

(2015):

 GLP()

. ( )
( )

/ .

. (Data )

.
.
.( )
.
.( )
/ .
.

 (3R)
() - IACUC
(Replacement)
:

(Reduction)
( )

( )
(Refinement)
(, , )

. 2013.

OECD

(OECD TG No. 423)

()

(OECD TG No. 420)

(OECD TG No. 425)
In vitro neutral red (OECD guidence 129)

(OECD TG No. 436)

(OECD TG No. 437)

EST-1000 (OECD TG No. 431)

SkinEthicTM (OECD TG No. 431)
Corrositex (OECD TG No. 435)
EpiSkinTM (OECD TG No. 431)
TER (OECD TG No. 430)

2 in vitro (OECD TG No. 439)

(OECD TG No. 439)

(LLNA) (OECD TG No. 429)

(rLLNA) (OECD TG No. 429)
-Brdu-Elisa (OECD TG No. 442B)
-DA (OECD TG No. 442A)

(EURL-EcVAM)
Bhas 42 (EURL-EcVAM)

()

(OECD TG No. 487)

alkaline Comet assay(OECD draft)

In vitro

(2015 in vitro EU )

Total animal No.; 1000

Beagle 50


2006; , NTP

2013; ,

2013; ,


2014; ,

2015; ,


-

13023 2015. 01. 20.

11987 2013. 07. 30.

KARA

()

()

23

 -HLS

 -


Historical data

//

3D
GLP

 -
In vivo In vitro
3D
3D bio printing

Organoid
Organoid

3D bio printing
3D bio printing


(Alternative to Animal Test Methods)

kmlim@ewha.ac.kr



3Rs (Russell and Burch,
1959)
Refine: painless test method
Reduce: reduce the number of animal use
Replace: replace with non-verterbrate organism, in
vitro , in chemico or in silico(QSAR) tests

(Animal Alternative Test)

3R ()
Replace ()

Regulations for the safe use of chemicals

The REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals)

Regulation.
The Biocidal Products Regulation (BPR); and also ongoing regulatory
responsibilities under the UK Control of Pesticides Regulations (COPR);
Plant Protection Products Directives and Regulations;
Detergents Regulations;
EU Classification, Labelling and Packaging Regulation
EU Cosmetic directives

Increases of animal sacrifice and costs by

implementation of REACH

The ECHA estimates that about 30,000 substances will be logged,

requiring 9 million animals to be used in tests costing 1.3 billion
(US$1.9 billion).
But a study by Hartung (Head of CAAT, Hopkins) suggests that this is
a gross underestimate, and that at least 68,000 chemicals will have to
be registered, requiring the use of 54 million animals (T. Hartung
and C. Rovida Nature 460, 10801081; 2009).

Impact of REACH on animal testing..

The total number of tests performed has increased (107 to 293)

A focus of REACH is to ensure a high level of protection of human

health and the environment, including the promotion of alternative
methods for assessment of hazards of substances (Chemworld 2014)

 ()
EU Cosmetic Directive 76/768/EEC (7th amendment
[2003/15/EC], summary from annual report 2008)

 ()
The sFDA recently issued a draft guideline for the in vitro 3T3 NRU
Phototoxicity assay as their first non-animal replacement (China sFDA,
2012b). Additionally, the sFDA has internally-issued a five-year program
on implementing alternatives into the review process along with a
training program (Personal Communication, February 2012).
On 4 February 2011, the MHLW, Japan was notified that data
obtained with alternative testing methods approved by the
Japanese Center for the Validation of Alternative Methods (JaCVAM)
could be used for the submission of quasi-drug applications, or for
petitions to include ingredients in the Standards for Cosmetics.

Additionally, administrative notices issued in April 2012 and May

2013 by the MHLW Evaluation and Licensing Division, publicised
the availability of alternative test methods for phototoxicity testing
and skin sensitisation testing for use in safety evaluations of
cosmetics and quasi-drugs.


The cosmetics industry has been strongly supporting the development
of alternative methods through the co-funding of the SEURAT-1 research
programme (2011-2015) together with the Commission.
Cosmetics Europe also invests in the promotion of the development, use
and regulatory acceptance of alternative methods globally. Different
international regulatory collaboration for networks are used as a vehicle,
including the International Cooperation on Cosmetic Regulations (ICCR).

Trends in the animal used in researches

Ban of Animal tests

EU Cosmetic Directive
REACH


Regulation for Cosmetics
EU (Dir 76/768/EEC)

KFDA ()

Acute toxicity (if available)

Single dose toxicity

Irritation and corrosion

Skin primary irritation test

, 3D skin model
Eye irritation test or other mucous mem (in vivo), BCOP& ICE (corr
brane irritation test
osion only)
Skin sensitization test
,
in vivo

Skin sensitization
Dermal/ percutaneous
Absorption
Repeated dose toxicity
Mutagenicity/ genotoxicity

Needed in case of bacteriocide or prese

rvative/sunscreen agent, tar color
Needed in case of bacteriocide or preserv
ative/sunscreen agent, tar color

Carcinogenicity

Reproductive toxicity

, in vivo

In vitro skin permeability

(Franz Cell)
, in vivo
AMES test, (in vitro)/
(in vivo)
(in vivo)

Needed in case of bacteriocide or preserv

ative/sunscreen agent, tar color

Toxicokinetics

(in vivo)

Photo-induced toxicity

Phototoxicity test/ photosensitization te

st but can be excepted when there is no ( in vivo, 3T3 NRU test)
UV absorption at UV range(280-420 nm)

Human data

Patch test
Cumulative patch test
Spraying agent or its ingredients

Inhalation toxicity

(GCP)

(Skin

(Eye

Irritation Test)

(Phototoxicity

irritation test)

(Skin sensitization

- (New

Test)

Zealand white

(Guinea

rabbit)

Pig)

test)/
(Photosensitizatio
n test)
or

 I
Chemical safety

Ethical Needs

: Skin primary irritation test (Rabbit)

:Eye irritation test (Rabbit Draize Test)

3D Human Skin Equivalent Model (Cytotoxicity Test)

3D Human Corneal Equivalent Model, Bovine Cornea Opacity/Permeability Test, Isolated Chick Eye Test.,
HET-CAM Test

: Skin sensitization test (Guinea Pig Maximization Test)

Dendritic cell models: h-CLAT(THP-1), U937 cells FACS analysis of surface markers.

In chemico: peptide reactivity assays/ c.f. Mouse local lymph node assay

: Phototoxicity Test (Guinea Pig Phototoxicity Test)

3T3 cell neutral red uptake test

 II


Going back to base and starting all over again ATLA 2011

Move out of the Comfort zone and find/build new and better methods !!

Outcome Mechanism-based ..
GLP 20 -

OECD Adverse Outcome Pathway

Adverse Outcome Pathway (AOP)

AOP for skin-sensitization

Example 1:
- Mechanism underlying Sensitization Test Methods

Induction Phase

Elicitation Reaction

NH2

p-Phenylenediamine
NH2

Haptenation:

GPMT, Buehler Test

NH2

NH2

DC
DC activation:

LNC Proliferation:

Example 1:
- Focusing on earlier stages of skin sensitization
Elicitation Reaction

Induction Phase
NH2

p-Phenylenediamine

ROS/Keap1:
Keratinosense

NH2

Haptenation:
Direct Peptide Reactivity Assay

NH2

NH2

DC
DC activation:
hCLAT assay
MUSST assay

LNC Proliferation:
Local Lymph Node
Assay

 II tool
biomedical or
FACS: Surface Marker - LLNA, hCLAT

Keratinosense Reporter gene assay: activation of Nrf II to Keap1 in MCF7

cells or Keratinocyte, Luciferase assay (Emter and Nasch et.al, Tox Appl.

Pharmacol. 2010)
LC/MSMS : Direct Peptide Reactivity Assay (DPRA)
ELISA: LLNA-BrdU-ELISA
RT-PCR: 21 gene expression profiles, Sensis (skin sensitization test)

 II tool - continued
human cell/tissue
Human cell-line: hCLAT (THP-1 cells), Human corneal cells (Eye irritation)
Human 3D Skin equivalent model (Epiderm, Episkin, OECD_TG_439) for Skin
irritation
EpiSkinTM

EpiDermTM

SkinEthic RHE Labcyte Epimodel

Human 3D cornea model (EpiOcular, HCE, MCTT HCE, Labcyte cornea

modeleit) for eye irritation (Jung et al., Tox in vitro. 2011, Jang et al., B&T 2015)

In chemico
- Direct peptide
reactivity assays

Artificial

Membrane
- Corrositex

Monolayer cell

Organotypic or

3D reconstructed

culture

Organ culture

human tissue

assay

model

Mammalian cells:
3T3 cells, MDCK,

- Bovine Cornea

U937 cells, Rabbit

Opacity/Permeabili

corneal cells

ty Test,

- Human cells:

- Isolated Chick

Human corneal

Eye Test.

cells

- HET-CAM

Epidermis, FullThickness Model

- Cornea Model

 III
(High Through-Put & Economic)
Well designed and scientifically validated alternative approaches :
are generally faster (than animal studies)
are generally cheaper
allow higher throughput

24, 96 well system

3D Tissue model (24, 48 and96 well)/
3T3 NRU Test (96 well)

Measurement of multiple endpoints with same number of animals

FACS analysis of multiple surface markers for profiling and cell-subtyping
LLNA:FCM Cell proliferation, B/T cell ration, ex vivo cytokine release (FACS,
ELISA, Jung et.al, Tox. Lett. 2012)

 IV Better prediction for human

toxicity
Well designed and scientifically validated alternative approaches :
provide equal or better prediction of human toxicity than the
corresponding animal test

Human is not a 70 kg rat!!

Concordance between animal and human toxicity

Nature reviews in drug discovery 2007

 V , , ,
, (Near in vivo, near human study)

: , ,
Cornea model

(Mol Pharmacol. 2008)

ADME :

Oral epithelium

- (PLoS ONE 2012)

- (Arch Drug Inf. 2011)
: proteomics,

Vaginal epithelium

metabolomics, genomics (Toxicol

In Vitro. 2006)

Pigmented skin

Skin-Ethics, LOreal & Mattek

MATTEK, USA

In near future, artificial tissues will replace all

human parts!!

Examples of Artificial Human Skin Models of Korean Origin

- KeraSkin, NeoDerm
KeraSkinVM,
Epidermis Model
, MCTT

NeoDerm, Full Thickness Model, TegoScience

Limitation of AATs. I
Systemic toxicity tests including reprotox, carcinogenicity, repeat dose
toxicity and toxicokinetic tests have no validated or endorsed AATs.
** 2011 Deadline (57 or , Sarah Adler et al, Arch Toxicol 2011)

Most AATs do not reach the level of regulatory tests.

** establish

SOP Background data (historical data)

REACH sees use of alternative test methods increasing but in

vivo tests more than double (Chemworld 2014)

But, problem and limitation of AATs...

Validation /
To validate an (alternative) test method is to establish the reliability and relevance

of the method for a particular purpose (Balls et al(1990). ATLA18,313-337 ,

OECD Guidance Document 34(2005))
*reliability: reproducibility of results (within and between laboratories and overtime)
*relevance: extent to which a test method correctly predicts the biological effect of
interest)


Research

Method
development
Method
Evaluation
Hazard
characterisation
Risk
assessment

Understanding the mechanisms through which chemicals can

induce a specific effect in humans

Developing non-animal test methods to predict chemicalinduced changes in each mechanistic pathway of interest

Evaluating the reliability (robust, reproducible & transferable) of

each non-animal test method using experimental controls

Evaluating the predictive capacity (in vitro/in chemico/in silico

to in vivo correlation) & applicability domain of each nonanimal test method
Evaluating the usefulness of toolbox of non-animal test methods for

predicting dose-response information in various exposure scenarios


Depending on the use, different levels of validation is necessary
Alternative methods for in-house purposes can be validated by the

company using the method..

Alternative methods intended to replace legally required animal studies,

undergo validation by an official validation body.

There is increasing collaboration between regional validation bodies

(ECVAM, ICCVAM, JACVAM and hopefully KoCVAM) to avoid
duplication of work and speed up consensus and harmonization (towards
OECD)


OECD guidelines for alternatives (Reduce or Replace)
OECD TG

Test

Replacing Test

TG430/431
/435

in vitro skin corrosion/human skin model

test/membrane barrier test

Acute dermal corrosion (TG404)

TG420/423
/425

In vivo acute dose toxicity-Fixed dose/Toxic

class method/up-and-down procedure

Acute dose toxicity-LD50

method (TG401)

TG428

Skin Absorption (in vitro)

Skin Absorption (In vivo) TG427

TG437

Bovine Corneal Opacity and Permeability

Test, Isolated chicken eye test for ocular
corrosion and severe irritation

Ocular corrosion TG405

TG429/442
A/442B/44
2C/442D

Skin sensitisation: Local Lymph Node

assay/DA/ELISA (in vivo), Direct peptide
reactivity assay, Keratinosens

Skin sensitisation (Guinea Pig)

TG406

TG432

In vitro 3T3 NRU phototoxicity Test

TG439

In vitro skin irritation: Reconstructed human

Acute dermal
irritation/corrosion (TG404)

TG 487

In vitro mammalian cell micronucleus test

Mammalian erythrocyte
micronucleus test TG474

epidermis test

 (AAT) 2015. 07

In vitro AAT
on validation
or replace AT in part
In vivo AAT
Available

Acute toxicity

In vitro AAT
Available

Photosensitisation

AAT
Not available

In vitro method

Status

Skin Corrosion

Human reconstituted skin

OECD TG431

Skin Irritation

Human reconstituted skin

OECD TG439

Phototoxicity

3T3 NRU

OECD TG432

Gene Toxicity

Ocular Toxicity
Acute Oral LD50

Micronucleus, Ames, Chromosome Aberrati OECD TG487, 471,

on, Mouse lymphoma TK assay

472, 473, 476

BCOP, ICE, microphysiometer

OECD437

Human reconstituted corneal model

OECD492

Acute Tox (CeeTox, Inc)

Prevalidation
?

Carcinogenecity
Skin Photosensitization

SenCeeTox -Photo (CeeTox, Inc)

Early Prevalidation

Skin sensitization

hCLAT, DPRA, KeratinosensTM

OECD442C/442D

Toxicokinetics

Reproductive Toxicity

Teratogenesis

Repeat Dose Systemic Toxicity Systemic Toxicity Panel (CeeTox, Inc)

Early Prevalidation

 Partial replacement
in 2014-2015 (TG442s)

Other approaches to reduce animal experiments

In vitro (test tube) test methods and models based on human cell and
tissue cultures
Computerized databases (QSAR, read-across) and virtual screenings with
computer models and simulations
Stem cell and genetic testing methods
Using humans
Non-invasive imaging techniques such as MRIs and CT Scans
microdosing (in which humans are given very low quantities of a drug to test the
effects on the body on the cellular level, without affecting the whole body system)

PETA




, ,
human-relevant
(
)

, , ,

Thanks for listening !!

Adverse Outcome Pathway

()
2015. 10. 28


The NAS/NRC Tox-21c report calls for a paradigm shift in toxicology

Toxicity Pathways
Animal testing wont disappear overnight, but the
agencies work signals the beginning of the end.
Elias Zerhouni, 15th Director of NIH

Toxicity testing was expensive, time-consuming, used

animals in large numbers, and didnt always work.
NAS/NRC 2007
Francis Collins, current Director of the NIH

Descriptive toxicology
in vivo animal models

Mechanistic toxicology
in vitro/silico models

Top-down development of
new toxicological tools

Bottom-up support to
alternative methods and legislative pressure

Critical Path Initiative

ToxCastTM

Tox21

US FDA

US EPA

US EPA, NIH, & FDA

EU-REACH

EU-REACH

Proposal

Regulation

Full ban animal testing

for cosmetics

Governmental efforts for alternatives to animal tests

JaCVAM
(Japanese Center for the
Validation of Alternative Methods)

ICCVAM
(Interagency Coordinating
Committee on the Validation of
Alternative Methods)

EURL ECVAM
(European Union Reference
Laboratory for alternatives to
animal testing)

KoCVAM
(Korean Center for the Validation
of Alternative Methods)

Current

Future

 -
/

(- )

, HTS

In vitro

physiology

OECD AOP development

Quoted from Dr. Donna L. Mendricks presentation at Adverse Outcome

Pathways: From Research to Regulation workshop 2014

Quoted from Dr. Donna L. Mendricks presentation at Adverse Outcome

Pathways: From Research to Regulation workshop 2014

Quoted from Stephen W. Edwardss presentation at Adverse Outcome

Pathways: From Research to Regulation workshop 2014

Quoted from Stephen W. Edwardss presentation at Adverse Outcome

Pathways: From Research to Regulation workshop 2014

Quoted from Stephen W. Edwardss presentation at Adverse Outcome

Pathways: From Research to Regulation workshop 2014

Example : AOP for skin sensitization

OECD Project 1.1 (Joanna Matheson, 2014)

https://aopkb.org/aopwiki/index.php/Skin_Sensitisation_Initiated_by_
Covalent_Binding_to_Proteins

OECD test guidelines

Direct peptide reactivity assay (DPRA)

KeratinoSens
Other tests
Human cell line activation test (h-CLAT)
Murine local lymph node assay(LLNA)

How to Develop an AOP

AOP-Wiki(http://aopwiki.org)

Five fundamental principles that guide AOP development

(1) AOPs are not chemical specific

(2) AOPs are modular and composed of reusable
components-notably key events (KEs) and key event
relationships (KERs)
(3) Individual AOP, composed of a single sequence of
KEs and KERs, is a pragmatic unit of AOP
development and evaluation
(4) Networks composed of multiple AOPs that share
common KEs and KERs are likely to be the functional
unit of prediction for most real-world scenarios
(5) AOPs are living documents that will evolve over time
as new knowledge is generated.

AOP Internal review charge questions

Section 1:AOP identifier/Title

Does the name of the AOP follow the right convention (MIE or first KE leading to AO)?
Does the name of the AOP reflect its content/domain?
Section 2: Authors
Is it clear who the authors/developers of the AOP are?
Contact information for one or more corresponding author(s) should be included.
Section 3: Date of updating
Reviewer should indicate the date stamp on the PDF snapshot under review.
Is it clear when the AOP was last updated?
Section 4: Abstract
Does the abstract concisely describe the main content of the AOP?
Section 5:
Molecular Initiating Event
Is a MIE described? If yes, then:
Is the MIE description clear covering biological state, biological compartment and role in the biology
and is it biologically plausible?
Is the MIE described in a way that allows its use in other AOPs?
Are measurement/prediction methods specified and adequately described/referenced? Is the biological context
(inc. taxonomic applicability/relevance, level of biological organisation) specified and explained sufficiently?
Have chemical initiators (prototypical chemicals or chemical features) been identified?
Key Events
Are the KE descriptions clear on how the events work covering biological state, biological compartment and role in the biolog
and are they biologically plausible?
Are the KEs described in a way that allows their reuse in other AOPs?
Are measurement methods specified and adequately described/referenced?
Is the biological context (inc. taxonomic applicability/relevance, level of biological organisation) specified
and explained sufficiently?

Adverse Outcome
Is an AO described? If yes, then:
Is the AO description clear and is it biologically plausible?
Is the AO described in a way that allows its use in other AOPs?
Are measurement methods specified and adequately described/referenced?
Is the biological context (inc. taxonomic applicability/relevance, level of biological organisation) specified
and explained sufficiently?
Has the regulatory relevance of the AO been described?
Section 6: Key Event Relationships
Are the KERs well described and in a way that allows their use in other AOPs?
Are the KERs biologically plausible and is there sufficient evidence presented?
''Is the level of empirical support adequately described in accordance with the OECD AOP Handbook?''
Are inconsistencies, uncertainties and level of confidence adequately described?
''Is the quantitative understanding of the KER described?"
Section 7: Overall Assessment of the AOP
Is the domain of applicability of the AOP defined appropriately?
Is the level of support for essentiality of the KEs adequately described and assessed?
Has the degree of quantitative understanding of KERs been assessed properly?
Has consideration been given to the overall weight of evidence for the AOP?
Are the calls on Overall WoE and Quantitative Understanding supported?
Section 8: Potential application of the AOP (optional):
Is any context provided as regards the reason for development or the intended use?
General Observations and Conclusions of the Reviewer

AOP under developing

 AOP
38

Finland
Switzerland

Norway
1

Belgium

Korea

10
4

United States
EPA, NIEHS, FDA

European
Commission
Canada

Ireland

Japan

United Kingdom

Last updated on August, 2015

Data resources : AOP wiki and OECD AOP list

24

1. Developing in KIT
Literature-based AOP in Hypothyroidism
Chemical
Class

Molecular
Initiating Event

Cellular
Response

KE

KE
Decreased
Thyroid
Hormone
Synthesis

Antithyroid drugs
Amiodarone
Lithium

Organ
Response

KE
w

Serum
-Decreased T4
-Increased Creatine

Rental tubule
Cytoplasmic
vacuolization
Cystic dilatation

Podocyte

Weight of evidence (WoE)

s

Strong

Weak

Cytoplasmic
vacuolization

KE
w

Decreased
glomerular
filtration

Decreased
renal plasma
flow
Decreased
sodium
reabsorption
Decreased renal
ability to dilute
urine

Montenegro J et al, 1996

Verhelst J et al, 1997
Karanikas et al, 2004
P Iglesias et al, 2009

Organism
Response

Kidney
Toxicity

2. Developing in KIT
Literature-based AOP in damage of lipid bilayer
Molecular
Initiating Event

Phospholipase A
inhibition

Cellular
Response

Damage of
lipid bilayer

Disturbance of
lysosomal
function

Mitochondrial
injury

Organism
Response

Organ
Response
Hepatocyte
Cytoplasmic
vacuolization
Ballooning
degeneration

Bile duct cell

Microvesicular
fat induction

Mallory body
formation

Cytoplasmic
vacuolization

Kupffer cell

Weight of evidence (WoE)

s

Strong

Weak

Cytoplasmic
vacuolization

Zimmerman HJ et al, 1999

Sampson KJ et al, 2011
De Marzio DH et al, 2013

Fibrosis

Thanks to
www.predictatox.org

Any questions or comments to:

sjyoon@kitox.re.kr

21st Century Toxicology and Medical Science:

Investing in Human Biology-Based Approaches of the Future

Importance of alternative test and its development,
cosmetics industry perspective
An Susun, Ph.D.
Amorepacific R&D Center
2015 Oct 28th

 ?
Methods for the determination of a biological (especially, toxicological)
endpoints that results in 3Rs of animal use but not the expense of human
safety
- Replacement: the substitution for conscious living higher animals of
insentient material.
- Reduction : reduction in the numbers of animal used to obtain information of a
given amount and precision.
- Refinement : any decrease in the incidence or severity of inhumane
procedures applied to those animals which still have to be used
*Russell and Burch (1959, in the Book of 'The principles of humane experimental technique'.)


Animal protection
-

Prohibition of animal test : animal welfare groups

Animal protection law : government - EU, Israel, India, Brazil

EU Cosmetic Directive
-

Animal testing and marketing bans finished products and prototypes from
September 2004.

Animal testing and marketing bans of ingredients from March 2009 (some exception)

Full marketing ban from March, 2013

EU REACH (Registration, Evaluation, Authorization and restriction of

Chemicals)
-

Requires all companies manufacturing or importing chemical substances into EU in

quantities of one tone or more per year to register these substances with the
European Chemicals Agency - For protection of human health and the environment

Need more safety data: Potential increase in animal testing and conflict with animal
testing ban for cosmetic ingredients - Accept the test results obtained using
validated alternative methods
3

: 3

2013.03

2014.11

: ,

2015.04

()

2013.01

: 2014 6
,

2014.01

2015.03

: , ,

, , ,

5 (2014.12)
-
- : , ,

(2015.03)

.
.

 (EU)
The Commission considers that animal testing that has clearly been motivated by
compliance with non-cosmetics related legislative frameworks should not be
considered to have been carried out 'in order to meet the requirements of this
Directive/Regulation'. The resulting animal testing data should not trigger the
marketing ban and could subsequently be relied on in the cosmetics safety
assessment. Reliance on such data is subject to its relevance for the cosmetics
safety assessment and its compliance with data quality requirements23.
Testing carried out for cosmetics relevant endpoints on ingredients that have been
specifically developed for cosmetic purposes and are exclusively used in cosmetic
products would in the Commission's view always be assumed to be carried out 'in
order to meet the requirements of this Directive/Regulation'.
The Commission considers that the marketing ban is triggered by the reliance on
the animal data for the safety assessment under the Cosmetics
Directive/Regulation, not by the testing as such. In case animal testing was carried
out for compliance with cosmetics requirements in third countries, this data
cannot be relied on in the Union for the safety assessment of cosmetics.
*COMMUNICATION FROM THE COMMISSION TO THE EUROPEAN PARLIAMENT AND THE COUNCIL on the animal testing
and marketing ban and on the state of play in relation to alternative methods in the field of cosmetics
(http://ec.europa.eu/consumers/archive/sectors/cosmetics/files/pdf/animal_testing/com_at_2013_en.pdf)


2
: ,

Council Directive 76/768/EEC (EU)

: Article 1. cosmetic product shall mean any substance or preparation
intended to be placed in contact with the various parts of the human body
(epidermis, hair system, nails, lips and external genital organs) or with
the teeth and the mucous membranes of the oral cavity with a view
exclusively or mainly to cleaning them, perfuming them, changing their
appearance and/or correcting body odours and/or protecting them or
keeping them in good condition
Article 2. cosmetic product must not cause damage to human health when

applied under normal or reasonably foreseeable conditions of use

6

- 2012 2 , negative list (

)
- 3
,

-
,

(EU)
-

 History
1997- 1999 : in vitro
6 , , ,
3 ( : ()).

2006 ~ : 2006 ()

2006. 01: KFDA AP

2006. 05, 07: Symposium

2007. 02: (Korean Society for Alternative to Animal
Experiments(KSAAE))
2007. 08: 6th World Congress on Alternatives and Animal Use in Life
Sciences ,
2009. 11: KoCVAM (Korean Center for the Validation of Alternative Methods)
8

HET-CAM for eye irritation tests

3T3 NRU assay for phototoxicity tests (OECD TG 432)
Skin senstization: DPRA (OECD TG 442C), Skin senstization: ARE-Nrf2(OECD TG
442D), h-CLAT (OECD draft TG)

Skin irritation test using 3D reconstructed human skin model (KeraskinTM) with
Korean skin tissues.
Eye irritation test using 3D reconstructed human corneal model from Korean
corneal tissues.
Oral mucosal irritation test using 3D reconstructed human oral mucous model from
Korean oral mucosal tissues

Integrated testing strategy (ITS) or tiered test with in vitro assays (skin sensitization,
eye irritation)
Appropriate use of human volunteer within the ethically permitted condition for
irritation test
9

 : ITS for sensitization

.

Skin penetration
Protein/peptide reactivity
: DPRA (TG 442C)
Cellular response of skin cells :
ARE-Nrf2(TG 442D), TG)
Functional activation of DCs :
h-CLAT (OECD draft

T cell proliferation

Computer based (Q)SAR

ConfirmHuman Repeat Insult Patch Test
Source : Report for establishing the timetable for phasing out animal testing for the purpose of the Cosmetics Directive chapter
3-4 skin sensitization http://ec.europa.eu/enterprise/cosmetics/doc/antest/(5)_chapter_3/4._skin_sensitisation_(final)_(1).pdf)

10

11

12




-
-

http://www.thevegetariansite.com/ethics_test.htm
13