국회토론회 발표자료 종합 korea general assembly forum: 21st century toxicology and...

152-154 Curtain Road, Shoreditch (EC2A 3AT

Troy SeidleDirector, Research & Toxicology [email protected]

Humane Society Internationalhsi.org/endanimaltesting

Advancing Safety Science and Health Research with Human Biology-

Based Tools of the 21st Century

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

About HSIHSI is the leading

international NGO

working to advance

non-animal safety

testing and bioscience

research worldwide.

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 regulatory science team representation

HSI research & toxicology portfolio worldwide

HSIs vision

Shift to a fully human biology-based paradigm based on understanding of adverse outcome pathways (AOPs)

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

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 & tradeAPRIL 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 morealternative methods available

Significant progress in development and validation of 3R testing tools (cell and computer models), as well as non-testingapproaches (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

2a. Evolving approaches to testing and assessment

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-nrf2luciferase), 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)

OECD guideline tests with replacement potential

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)

Acceptance of available alternatives in regulation

Ongoing 3R revisions to EU REACH data requirements

HSI suggestions for updating K-REACH requirementsEndpoint 3R best practice EU KR

Reproductive toxicity

Extended 1-generation study (EOGRTS) instead of 2-gen (- 1,200 animals = 46% reduction)

Genetic toxicity, micronucleus

Assess in combination with a repeated dose toxicity study rather than 2 separate studies (- 80 animals)

Acute toxicity, dermal

Waive study requirement if substance is unclassified (non-toxic) via oral route (- 30 animals)

Carcinogenicity 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 availablealternatives

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 2014-20 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 Congress15-18 November 2016, Fukuoka, JapanKorea represented on program committee

Encouraging increased Korean involvement in the OECD global research effort to map human AOPs

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

3. Toward a 21st century medical research paradigm

Toward a 21st century medical research paradigmRallying 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 SeidleDirectorResearch & Toxicology [email protected]

Borami SeoPolicy Advisor, Research & Toxicology DepartmentSeoul, [email protected]

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

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 .

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

21.2.2015 L 50/1 Official Journal of the European Union EN

(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.

Troy Seidle

Troy Seidle

(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 developmental 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).

(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


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 necessary. However, testing for developmental toxicity must be considered.

If a substance is known to cause developmental toxicity, meeting the criteria 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 developmental 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 Reproductive Toxicity Study (Annex IX, section 8.7.3) or a pre-natal developmental 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 Reproductive Toxicity Study (B.56 of the Commission Regulation on test methods as specified in Article 13(3) or OECD 443), basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure, if the available repeated dose toxicity studies (e.g. 28-day or 90-day studies, OECD 421 or 422 screening studies) indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

8.7.3. An Extended One-Generation Reproductive Toxicity Study with the extension of cohort 1B to include the F2 generation shall be proposed by the registrant or may be required by the Agency in accordance with Article 40 or 41, if: (a) the substance has uses leading to significant exposure of

consumers or professionals, taking into account, inter alia, consumer exposure from articles, and

(b) any of the following conditions are met:

the substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or

there are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, 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.


An Extended One-Generation Reproductive Toxicity Study including 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 accordance with Article 40 or 41, in case of particular concerns on (developmental) neurotoxicity or (developmental) immunotoxicity 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 structurally analogous to the substance being studied, suggesting such effects or mechanisms/modes of action.

Other studies on developmental neurotoxicity and/or developmental immunotoxicity instead of cohorts 2A/2B (developmental neurotoxicity) and/or cohort 3 (developmental immunotoxicity) 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 perform 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 Reproductive Toxicity Study (B.56 of the Commission Regulation on test methods as specified in Article 13(3) or OECD 443), basic test design (cohorts 1A and 1B without extension to include a F2 generation), one species, most appropriate route of administration, having regard to the likely route of human exposure, unless already provided as part of Annex IX requirements.

8.7.3. An Extended One-Generation Reproductive Toxicity Study with the extension of cohort 1B to include the F2 generation shall be proposed by the registrant or may be required by the Agency in accordance with Article 40 or 41, if: (a) the substance has uses leading to significant exposure of

consumers or professionals, taking into account, inter alia, consumer exposure from articles, and

(b) any of the following conditions are met:

the substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or

there are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, 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.


An Extended One-Generation Reproductive Toxicity Study including 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 accordance with Article 40 or 41, in case of particular concerns on (developmental) neurotoxicity or (developmental) immunotoxicity 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 structurally analogous to the substance being studied, suggesting such effects or mechanisms/modes of action.

Other studies on developmental neurotoxicity and/or developmental immunotoxicity instead of cohorts 2A/2B (developmental neurotoxicity) and/or cohort 3 (developmental immunotoxicity) 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.


EN EN

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


Troy Seidle

EN 2 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


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.

1 OJ L 396, 30.12.2006, p. 1. 2 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).

3 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 3 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 Co-operation 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.

4 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 4 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)

1

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 "


"8.2. Serious eye damage/eye

irritation

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.

8.2.1 Serious eye damage/

eye irritation, in vitro"

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.

2


"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 first-

choice method for in vivo testing. Only in exceptional

circumstances should another test be used. Justification for

the use of another test shall be provided."

3

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)."


"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."


"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

4

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):

?

. ( )

( )

/ .

.

.

. (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

2013; ,

2006; , NTP

2013; ,

2014; ,

2015; ,

11987 2013. 07. 30.

13023 2015. 01. 20.

-

-

KARA

()

() 23

-

-

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)

[email protected]

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 ()

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

Regulations for the safe use of chemicals

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).

Increases of animal sacrifice and costs by implementation of REACH

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)

Impact of REACH on animal testing..

()

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 , in vivo

Irritation and corrosion Skin primary irritation test

Eye irritation test or other mucous mem

brane irritation test

, 3D skin model (in vivo), BCOP& ICE (corrosion only)

Skin sensitization Skin sensitization test , in vivo

Dermal/ percutaneous

Absorption

In vitro skin permeability

(Franz Cell)

Repeated dose toxicity Needed in case of bacteriocide or prese

rvative/sunscreen agent, tar color

, in vivo

Mutagenicity/ genotoxicity Needed in case of bacteriocide or preserv

ative/sunscreen agent, tar color

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

Carcinogenicity (in vivo)

Reproductive toxicity 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

UV absorption at UV range(280-420 nm)

( in vivo, 3T3 NRU test)

Human data Patch test

Cumulative patch test

(GCP)

Inhalation toxicity Spraying agent or its ingredients

(Skin

Irritation Test)

- (New

Zealand white

rabbit)

(Phototoxicity

Test)

(Guinea

Pig)

(Eye

irritation test)

(Skin sensitization

test)/

(Photosensitizatio

n test)

or

I

Chemical safety

Ethical Needs

: Skin primary irritation test (Rabbit)

3D Human Skin Equivalent Model (Cytotoxicity Test)

:Eye irritation test (Rabbit Draize 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

Induction Phase

NH2

NH2

T

DC

NH2

NH2

Elicitation Reaction

p-Phenylenediamine

Example 1: - Mechanism underlying Sensitization Test Methods

Haptenation:

DC activation: LNC Proliferation:

GPMT, Buehler Test

Induction Phase

NH2

NH2

T

DC

NH2

NH2

Elicitation Reaction

p-Phenylenediamine

Example 1: - Focusing on earlier stages of skin sensitization

Haptenation: Direct Peptide Reactivity Assay

DC activation:hCLAT assayMUSST assay

LNC Proliferation:Local Lymph NodeAssay

ROS/Keap1: Keratinosense

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

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)

EpiSkinTM EpiDermTM SkinEthic RHE Labcyte Epimodel

In chemico

- Direct peptide

reactivity assays

Artificial

Membrane

- Corrositex

Monolayer cell

culture

Mammalian cells:

3T3 cells, MDCK,

U937 cells, Rabbit

corneal cells

- Human cells:

Human corneal

cells

Organotypic or

Organ culture

assay

- Bovine Cornea

Opacity/Permeabili

ty Test,

- Isolated Chick

Eye Test.

- HET-CAM

3D reconstructed

human tissue

model

Epidermis, Full-

Thickness 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

Concordance between animal and human toxicity Nature reviews in drug discovery 2007

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!!

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

Cornea model

Oral epithelium

Pigmented skin

Vaginal epithelium

: , ,

(Mol Pharmacol. 2008)

ADME :

- (PLoS ONE 2012)

- (Arch Drug Inf. 2011)

: proteomics,

metabolomics, genomics (Toxicol

In Vitro. 2006)

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 (5-

7 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)

Understanding the mechanisms through which chemicals can

induce a specific effect in humansResearch

Developing non-animal test methods to predict chemical-

induced changes in each mechanistic pathway of interest

Method

development

Evaluating the reliability (robust, reproducible & transferable) of

each non-animal test method using experimental controls

Method

Evaluation

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

to in vivo correlation) & applicability domain of each non-

animal test method

Hazard

characterisation

Evaluating the usefulness of toolbox of non-animal test methods for

predicting dose-response information in various exposure scenariosRisk

assessment

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/Toxicclass 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

TG429/442A/442B/442C/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 epidermis test

Acute dermal irritation/corrosion (TG404)

TG 487 In vitro mammalian cell micronucleus test Mammalian erythrocyte micronucleus test TG474

Acute toxicity

AAT

Not available

Photosensitisation

In vitro AAT

Available

(AAT) 2015. 07

In vivo AAT

Available

In vitro AAT

on validation

or replace AT in part

In vitro method Status

Skin Corrosion Human reconstituted skin OECD TG431

Skin Irritation Human reconstituted skin OECD TG439

Phototoxicity 3T3 NRU OECD TG432

Gene ToxicityMicronucleus, Ames, Chromosome Aberrati

on, Mouse lymphoma TK assay

OECD TG487, 471,

472, 473, 476

Ocular ToxicityBCOP, ICE, microphysiometer

Human reconstituted corneal model

OECD437

OECD492

Acute Oral LD50 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)

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)

Other approaches to reduce animal experiments

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

Toxicity testing was expensive, time-consuming, used

animals in large numbers, and didnt always work.

Animal testing wont disappear overnight, but the

agencies work signals the beginning of the end.

Descriptive toxicologyin vivo animal models

Mechanistic toxicologyin vitro/silico models

Elias Zerhouni, 15th Director of NIH

Francis Collins, current Director of the NIH

NAS/NRC 2007

Top-down development of

new toxicological tools

Bottom-up support to

alternative methods and legislative pressure

Critical Path InitiativeUS FDA

EU-REACH Proposal

EU-REACHRegulation

ToxCastTM

US EPA

Tox21US EPA, NIH, & FDA

Full ban animal testing

for cosmetics

Governmental efforts for alternatives to animal tests

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)

JaCVAM

(Japanese 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 Stephen W. Edwardss presentation at Adverse Outcome Pathways: From Research to Regulation workshop 2014

Example : AOP for skin sensitization

OECD test guidelines Direct peptide reactivity assay (DPRA) KeratinoSens Other tests Human cell line activation test (h-CLAT) Murine local lymph node assay(LLNA)

OECD Project 1.1 (Joanna Matheson, 2014)https://aopkb.org/aopwiki/index.php/Skin_Sensitisation_Initiated_by_Covalent_Binding_to_Proteins

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.

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 biology

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?

AOP Internal review charge questions

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

European

Commission

United States

EPA, NIEHS, FDA

Japan

Canada

Switzerland

United Kingdom

Norway

Belgium

38

5

3

5

10

1

42

AOP

24

Korea

2

Finland

Ireland

1

1

Last updated on August, 2015Data resources : AOP wiki and OECD AOP list

1. Developing in KITLiterature-based AOP in Hypothyroidism

ChemicalClass

MolecularInitiating Event

OrganResponse

OrganismResponse

Decreased Thyroid

HormoneSynthesis

-Decreased T4 -Increased Creatine

Decreased glomerular filtration

Decreased renal plasma

flow

Decreased sodium

reabsorption

Decreased renal ability to dilute

urine

Kidney Toxicity

SerumAntithyroid drugs

Amiodarone Lithium

Rental tubule

Cytoplasmic vacuolizationCystic dilatation

Podocyte

Cytoplasmicvacuolization

CellularResponse

KE KE

Strong

Weak

s

w

Weight of evidence (WoE)

s sw w

KE KE

Montenegro J et al, 1996Verhelst J et al, 1997Karanikas et al, 2004P Iglesias et al, 2009

2. Developing in KITLiterature-based AOP in damage of lipid bilayer

Damage of lipid bilayer

FibrosisPhospholipase A inhibition

Disturbance of lysosomal function

Mitochondrial injury

Bile duct cell

Cytoplasmic vacuolization

Hepatocyte

Cytoplasmic vacuolization Ballooning

degeneration

Cytoplasmic vacuolization

Microvesicular fat induction

Mallory body formation

MolecularInitiating Event

OrganResponse

OrganismResponse

CellularResponse

Kupffer cell

Strong

Weak

s

w

Weight of evidence (WoE)

s sw w

Zimmerman HJ et al, 1999Sampson KJ et al, 2011De Marzio DH et al, 2013

Thanks to

www.predictatox.org

Any questions or comments to:

[email protected]

An Susun, Ph.D.

Amorepacific R&D Center

2015 Oct 28th

1

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

?

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'.)

2

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

4

: 3

: ,

: 2014 6

,

: , ,

, , ,

()

2013.032014.11

2013.01

2014.01

2015.03

2015.04

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)

5

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)

-

7

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

.

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)

Skin penetration

Protein/peptide reactivity : DPRA (TG 442C)

T cell proliferation

Computer based (Q)SAR Human Repeat Insult Patch Test

Cellular response of skin cells : ARE-Nrf2(TG 442D), TG) Functional activation of DCs : h-CLAT (OECD draft

Confirm

10

-

-

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

13

0. 1. HSI Korea GA mtg_final1-1. 1-2. CELEX_32015R0282_EN_TXTCOMMISSION 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)

1-3. EU317_EN_1_11-4. EU317_EN_2_12. _3. 2015___4. _AOP__final5. 10 __1027

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