TRANSGENIC RODENT SOMATIC AND GERM CELL GENE MUTATION ASSAYS: RETROSPECTIVE PERFORMANCE ASSESSMENT

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1 Unclassified English - Or. English Unclassified ENV/JM/MONO(2011)20 Organisation de Coopération et de Développement Économiques Organisation for Economic Co-operation and Development 18-Aug-2011 English - Or. English ENVIRONMENT DIRECTORATE JOINT MEETING OF THE CHEMICALS COMMITTEE AND THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY TRANSGENIC RODENT SOMATIC AND GERM CELL GENE MUTATION ASSAYS: RETROSPECTIVE PERFORMANCE ASSESSMENT Series on Testing and Assessment No. 145 JT Document complet disponible sur OLIS dans son format d'origine Complete document available on OLIS in its original format

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3 OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 145 TRANSGENIC RODENT SOMATIC AND GERM CELL GENE MUTATION ASSAYS: RETROSPECTIVE PERFORMANCE ASSESSMENT Environment Directorate ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT Paris

4 Also published in the Series on Testing and Assessment: No. 1, Guidance Document for the Development of OECD Guidelines for Testing of Chemicals (1993; reformatted 1995, revised 2006) No. 2, Detailed Review Paper on Biodegradability Testing (1995) No. 3, Guidance Document for Aquatic Effects Assessment (1995) No. 4, Report of the OECD Workshop on Environmental Hazard/Risk Assessment (1995) No. 5, Report of the SETAC/OECD Workshop on Avian Toxicity Testing (1996) No. 6, Report of the Final Ring-test of the Daphnia magna Reproduction Test (1997) No. 7, Guidance Document on Direct Phototransformation of Chemicals in Water (1997) No. 8, Report of the OECD Workshop on Sharing Information about New Industrial Chemicals Assessment (1997) No. 9, Guidance Document for the Conduct of Studies of Occupational Exposure to Pesticides during Agricultural Application (1997) No. 10, Report of the OECD Workshop on Statistical Analysis of Aquatic Toxicity Data (1998) No. 11, Detailed Review Paper on Aquatic Testing Methods for Pesticides and industrial Chemicals (1998) No. 12, Detailed Review Document on Classification Systems for Germ Cell Mutagenicity in OECD Member Countries (1998) No. 13, Detailed Review Document on Classification Systems for Sensitising Substances in OECD Member Countries 1998) No. 14, Detailed Review Document on Classification Systems for Eye Irritation/Corrosion in OECD Member Countries (1998) No. 15, Detailed Review Document on Classification Systems for Reproductive Toxicity in OECD Member Countries (1998) No. 16, Detailed Review Document on Classification Systems for Skin Irritation/Corrosion in OECD Member Countries (1998) 4

5 No. 17, Environmental Exposure Assessment Strategies for Existing Industrial Chemicals in OECD Member Countries (1999) No. 18, Report of the OECD Workshop on Improving the Use of Monitoring Data in the Exposure Assessment of Industrial Chemicals (2000) No. 19, Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals used in Safety Evaluation (1999) No. 20, Revised Draft Guidance Document for Neurotoxicity Testing (2004) No. 21, Detailed Review Paper: Appraisal of Test Methods for Sex Hormone Disrupting Chemicals (2000) No. 22, Guidance Document for the Performance of Out-door Monolith Lysimeter Studies (2000) No. 23, Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000) No. 24, Guidance Document on Acute Oral Toxicity Testing (2001) No. 25, Detailed Review Document on Hazard Classification Systems for Specifics Target Organ Systemic Toxicity Repeated Exposure in OECD Member Countries (2001) No. 26, Revised Analysis of Responses Received from Member Countries to the Questionnaire on Regulatory Acute Toxicity Data Needs (2001) No 27, Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment (2001) No 28, Guidance Document for the Conduct of Skin Absorption Studies (2004) No 29, Guidance Document on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (2001) No 30, Detailed Review Document on Hazard Classification Systems for Mixtures (2001) No 31, Detailed Review Paper on Non-Genotoxic Carcinogens Detection: The Performance of In-Vitro Cell Transformation Assays (2007) No. 32, Guidance Notes for Analysis and Evaluation of Repeat- Dose Toxicity Studies (2000) 5

6 No. 33, Harmonised Integrated Classification System for Human Health and Environmental Hazards of Chemical Substances and Mixtures (2001) No. 34, Guidance Document on the Development, Validation and Regulatory Acceptance of New and Updated Internationally Acceptable Test Methods in Hazard Assessment (2005) No. 35, Guidance notes for analysis and evaluation of chronic toxicity and carcinogenicity studies (2002) No. 36, Report of the OECD/UNEP Workshop on the use of Multimedia Models for estimating overall Environmental Persistence and long range Transport in the context of PBTS/POPS Assessment (2002) No. 37, Detailed Review Document on Classification Systems for Substances Which Pose an Aspiration Hazard (2002) No. 38, Detailed Background Review of the Uterotrophic Assay Summary of the Available Literature in Support of the Project of the OECD Task Force on Endocrine Disrupters Testing and Assessment (EDTA) to Standardise and Validate the Uterotrophic Assay (2003) No. 39, Guidance Document on Acute Inhalation Toxicity Testing (in preparation) No. 40, Detailed Review Document on Classification in OECD Member Countries of Substances and Mixtures Which Cause Respiratory Tract Irritation and Corrosion (2003) No. 41, Detailed Review Document on Classification in OECD Member Countries of Substances and Mixtures which in Contact with Water Release Toxic Gases (2003) No. 42, Guidance Document on Reporting Summary Information on Environmental, Occupational and Consumer Exposure (2003) No. 43, Guidance Document on Mammalian Reproductive Toxicity Testing and Assessment (2008) No. 44, Description of Selected Key Generic Terms Used in Chemical Hazard/Risk Assessment (2003) No. 45, Guidance Document on the Use of Multimedia Models for Estimating Overall Environmental Persistence and Long-range Transport (2004) No. 46, Detailed Review Paper on Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances (2004) 6

7 No. 47, Detailed Review Paper on Fish Screening Assays for the Detection of Endocrine Active Substances (2004) No. 48, New Chemical Assessment Comparisons and Implications for Work Sharing (2004) No. 49, Report from the Expert Group on (Quantitative) Structure- Activity Relationships [(Q)SARs] on the Principles for the Validation of (Q)SARs (2004) No. 50, (2005) Report of the OECD/IPCS Workshop on Toxicogenomics No. 51, Approaches to Exposure Assessment in OECD Member Countries: Report from the Policy Dialogue on Exposure Assessment in June 2005 (2006) No. 52, Comparison of emission estimation methods used in Pollutant Release and Transfer Registers (PRTRs) and Emission Scenario Documents (ESDs): Case study of pulp and paper and textile sectors (2006) No. 53, Guidance Document on Simulated Freshwater Lentic Field Tests (Outdoor Microcosms and Mesocosms) (2006) No. 54, Current Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application (2006) No. 55, Detailed Review Paper on Aquatic Arthropods in Life Cycle Toxicity Tests with an Emphasis on Developmental, Reproductive and Endocrine Disruptive Effects (2006) No. 56, Guidance Document on the Breakdown of Organic Matter in Litter Bags (2006) No. 57, Detailed Review Paper on Thyroid Hormone Disruption Assays (2006) No. 58, Report on the Regulatory Uses and Applications in OECD Member Countries of (Quantitative) Structure-Activity Relationship [(Q)SAR] Models in the Assessment of New and Existing Chemicals (2006) No. 59, Report of the Validation of the Updated Test Guideline 407: Repeat Dose 28-Day Oral Toxicity Study in Laboratory Rats (2006) No. 60, Report of the Initial Work Towards the Validation of the 21- Day Fish Screening Assay for the Detection of Endocrine Active Substances (Phase 1A) (2006) 7

8 No. 61, Report of the Validation of the 21-Day Fish Screening Assay for the Detection of Endocrine Active Substances (Phase 1B) (2006) No. 62, Final OECD Report of the Initial Work Towards the Validation of the Rat Hershberger Assay: Phase-1, Androgenic Response to Testosterone Propionate, and Anti-Androgenic Effects of Flutamide (2006) No. 63, Guidance Document on the Definition of Residue (2006) No. 64, Guidance Document on Overview of Residue Chemistry Studies (2006) No. 65, OECD Report of the Initial Work Towards the Validation of the Rodent Uterotrophic Assay - Phase 1 (2006) No. 66, OECD Report of the Validation of the Rodent Uterotrophic Bioassay: Phase 2. Testing of Potent and Weak Oestrogen Agonists by Multiple Laboratories (2006) No. 67, Additional data supporting the Test Guideline on the Uterotrophic Bioassay in rodents (2007) No. 68, Summary Report of the Uterotrophic Bioassay Peer Review Panel, including Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the follow up of this report (2006) No. 69, Guidance Document on the Validation of (Quantitative) Structure-Activity Relationship [(Q)SAR] Models (2007) No. 70, Report on the Preparation of GHS Implementation by the OECD Countries (2007) No. 71, Guidance Document on the Uterotrophic Bioassay - Procedure to Test for Antioestrogenicity (2007) No. 72, Guidance Document on Pesticide Residue Analytical Methods (2007) No. 73, Report of the Validation of the Rat Hershberger Assay: Phase 3: Coded Testing of Androgen Agonists, Androgen Antagonists and Negative Reference Chemicals by Multiple Laboratories. Surgical Castrate Model Protocol (2007) No. 74, Detailed Review Paper for Avian Two-generation Toxicity Testing (2007) No. 75, Guidance Document on the Honey Bee (Apis Mellifera L.) Brood test Under Semi-field Conditions (2007) 8

9 No. 76, Final Report of the Validation of the Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances: Phase 1 - Optimisation of the Test Protocol (2007) No. 77, Final Report of the Validation of the Amphibian Metamorphosis Assay: Phase 2 - Multi-chemical Interlaboratory Study (2007) No. 78, Final Report of the Validation of the 21-day Fish Screening Assay for the Detection of Endocrine Active Substances. Phase 2: Testing Negative Substances (2007) No. 79, Validation Report of the Full Life-cycle Test with the Harpacticoid Copepods Nitocra Spinipes and Amphiascus Tenuiremis and the Calanoid Copepod Acartia Tonsa - Phase 1 (2007) No. 80, Guidance on Grouping of Chemicals (2007) No. 81, Summary Report of the Validation Peer Review for the Updated Test Guideline 407, and Agreement of the Working Group of National Coordinators of the Test Guidelines Programme on the follow-up of this report (2007) No. 82, (2007) Guidance Document on Amphibian Thyroid Histology No. 83, Summary Report of the Peer Review Panel on the Stably Transfected Transcriptional Activation Assay for Detecting Estrogenic Activity of Chemicals, and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007) No. 84, Report on the Workshop on the Application of the GHS Classification Criteria to HPV Chemicals, 5-6 July Bern Switzerland (2007) No. 85, Report of the Validation Peer Review for the Hershberger Bioassay, and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2007) No. 86, Report of the OECD Validation of the Rodent Hershberger Bioassay: Phase 2: Testing of Androgen Agonists, Androgen Antagonists and a 5 α-reductase Inhibitor in Dose Response Studies by Multiple Laboratories (2008) No. 87, Report of the Ring Test and Statistical Analysis of Performance of the Guidance on Transformation/Dissolution of Metals and Metal Compounds in Aqueous Media (Transformation/ Dissolution Protocol) (2008) 9

10 No.88, Workshop on Integrated Approaches to Testing and Assessment (2008) No.89, Retrospective Performance Assessment of the Test Guideline 426 on Developmental Neurotoxicity (2008) No.90, Background Review Document on the Rodent Hershberger Bioassay (2008) No.91, Report of the Validation of the Amphibian Metamorphosis Assay (Phase 3) (2008) No.92, Report of the Validation Peer Review for the Amphibian Metamorphosis Assay and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-Up of this Report (2008) No.93, Report of the Validation of an Enhancement of OECD TG 211: Daphnia Magna Reproduction Test (2008) No.94, Report of the Validation Peer Review for the 21-Day Fish Endocrine Screening Assay and Agreement of the Working Group of the National Coordinators of the Test Guidelines Programme on the Follow-up of this Report (2008) No.95, Detailed Review Paper on Fish Life-Cycle Tests (2008) No.96, Guidance Document on Magnitude of Pesticide Residues in Processed Commodities (2008) No.97, Detailed Review Paper on the use of Metabolising Systems for In Vitro Testing of Endocrine Disruptors (2008) No. 98, Considerations Regarding Applicability of the Guidance on Transformation/Dissolution of Metals Compounds in Aqueous Media (Transformation/Dissolution Protocol) (2008) No. 99, Comparison between OECD Test Guidelines and ISO Standards in the Areas of Ecotoxicology and Health Effects (2008) No.100, (2009) Report of the Second Survey on Available Omics Tools No.101, Report of the Workshop on Structural Alerts for the OECD (Q)SAR Application Toolbox, May 2008, Utrecht, the Netherlands (2009) No. 102, Guidance Document for using the OECD (Q)SAR Application Toolbox to Develop Chemical Categories According to the OECD Guidance on Grouping of Chemicals (2009) 10

11 No. 103, Detailed Review Paper on Transgenic Rodent Mutation Assays (2009) No. 104, Performance Assessment: Conparsion of 403 and CxT Protocols via Simulation and for Selected Real Data Sets (2009) No. 105, Report on Biostatistical Performance Assessment of the draft TG 436 Acute Toxic Class Testing Method for Acute Inhalation Toxicity (2009) No. 106, Guidance Document for Histologic Evaluation of Endocrine and Reproductive Test in Rodents (2009) No. 107, Preservative treated wood to the environment for wood held in storage after treatment and for wooden commodities that are not cover and are not in contact with ground. (2009) No. 108, Report of the validation of the Hershberger Bioassay (weanling model) (2009) No. 109, Literature review on the 21-Day Fish Assay and the Fish Short-Term Reproduction Assay (2009) No. 110, Report of the validation peer review for the weanling Hershberger Bioassay and agreement of the working of national coordinators of the test guidelines programme on the follow-up of this report (2009) No. 111, Report of the Expert Consultation to Evaluate an Estrogen Receptor Binding Affinity Model for Hazard Identification (2009) No. 112, (2009) The 2007 OECD List of High Production Volume Chemicals No. 113, Report of The Focus Session on Current and Forthcoming Approaches for Chemical Safety and Animal Welfare (2010) No. 114, Performance Assessment of Different Cytotoxic and Cytostatic Measures for the In Vitro Micronucleus Test (MNVIT): Summary of results in the collaborative trial (2010) No. 115, Guidance Document on the Weanling Hershberger Bioassay in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties (2009) No. 116, Guidance Document on the Design and Conduct of Chronic Toxicity and Carcinogenicity Studies, Supporting TG 451, 452 and 453 (2010) No. 117, Guidance Document 117 on the Current Implementation of Internal Triggers in Test Guideline 443 for an Extended One 11

12 Generation Reproductive Toxicity Study, in the United States and Canada (2011) No. 118, Workshop Report on OECD Countries Activities Regarding Testing, Assessment and Management of Endocrine Disrupters Part I and Part II (2010) No. 119, Classification and Labelling of chemicals according to the UN Globally Harmonized System: Outcome of the Analysis of Classification of Selected Chemicals listed in Annex III of the Rotterdam Convention (2010) No. 120, Part 1: Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry Mechanism-based Structural Alerts for the Identification of DNA Binding Chemicals No. 120, Part 2: Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry Mechanism-based Structural Alerts for the Identification of DNA Binding Chemicals No. 121, Detailed review paper (DRP) on Molluscs life-cycle Toxicity Testing (2010) No. 122, Guidance Document on the determination of the Toxicity of a Test Chemical to the Dung Beetle Aphodius Constans (2010) No. 123, Guidance Document on the Diagnosis of Endocrine-related Histopathology in Fish Gonads (2010) No. 124, (2010) Guidance for the Derivation of an Acute Reference Dose No. 125, Guidance Document on Histopathology for Inhalation Toxicity Studies, Supporting TG 412 (Subacute Inhalation Toxicity: 28- Day) and TG 413 (Subchronic Inhalation Toxicity: 90-Day) (2010) No. 126, Short Guidance on the Threshold approach for Acute Fish Toxicity (2010) No. 127, Peer Review Report of the Validation of the 21-day Androgenised Female Stickleback Screening Assay (2010) No. 128, Validation Report of the 21-day Androgenised Female Stickleback Screening Assay (2010) No. 129, Guidance Document on using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests No. 131, Report of the Test Method Validation of Avian Acute Oral Toxicity Test (OECD test guideline 223) (2010) 12

13 No. 132, Report of the Multi-Laboratory Validation of the H295R Steroidogenesis Assay to Identify Modulators (2010) No.133, Peer Review Report for the H295R Cell-Based Assay for Steroidogenesis (2010) No.134, Report of the Validation of a Soil Bioaccumulation Test with Terrestrial Oligochaetes by an International Ring Test (2010) No.135, Detailed Review Paper on Environmental Endocrine Disruptor Screening: The use of Estrogen and Androgen Receptor Binding and Transactivation Assays in Fish (2010) No. 136, Validation Report of The Chironomid Full Life-Cycle Toxicity Test (2010) No. 137, Explanatory Background Document to the OECD Test Guideline On In Vitro Skin Irritation Testing (2010) No. 138, Report of the Workshop on Using Mechanistic Information in Forming Chemical Categories (2011) No. 139, Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry Mechanism Based Structural Alerts for the Identification of Protein-binding Chemicals (2011) No. 140, Report of the WHO/OECD/ILSI (Hesi) Workshop on Risk Assessment of Combined Exposures to Multiple Chemicals (2011) No. 141, Validation Report (Phase 1) for the Fish Sexual Development Test for the Detection of Endocrine Active Substances (2011) No. 142, Validation Report (Phase 2) for the Fish Sexual Development Test for the Detection of Endocrine Active Substances (2011) No. 143, Peer Review Report for the Validation of the Fish Sexual Development Test and Agreement of the Working Group of National Co-ordinators of the Test Guideline Programme on the Follow-up of the Peer Review (2011) No. 144, Validation report supporting the Test Guideline for the Acute Chironomid Assay (2011) 13

14 No. 145, Transgenic Rodent Somatic and Germ Cells Gene Mutation Assays: Retrospective Performance Assessment (2011) OECD 2011 Applications for permission to reproduce or translate all or part of this material should be made to: Head of Publications Service, OECD, 2 rue André-Pascal, Paris Cedex 16, France 14

15 ABOUT THE OECD The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental organisation in which representatives of 34 industrialised countries in North and South America, Europe and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise policies, discuss issues of mutual concern, and work together to respond to international problems. Most of the OECD s work is carried out by more than 200 specialised committees and working groups composed of member country delegates. Observers from several countries with special status at the OECD, and from interested international organisations, attend many of the OECD s workshops and other meetings. Committees and working groups are served by the OECD Secretariat, located in Paris, France, which is organised into directorates and divisions. The Environment, Health and Safety Division publishes free-of-charge documents in ten different series: Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides and Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety of Novel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission Scenario Documents; and Safety of Manufactured Nanomaterials. More information about the Environment, Health and Safety Programme and EHS publications is available on the OECD s World Wide Web site ( This publication was developed in the IOMC context. The contents do not necessarily reflect the views or stated policies of individual IOMC Participating Organisations. The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was established in 1995 following recommendations made by the 1992 UN Conference on Environment and Development to strengthen co-operation and increase international co-ordination in the field of chemical safety. The Participating Organisations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO, World Bank and OECD. UNDP is an observer. The purpose of the IOMC is to promote co-ordination of the policies and activities pursued by the Participating Organisations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. 15

16 This publication is available electronically, at no charge. For this and many other Environment, Health and Safety publications, consult the OECD s World Wide Web site ( or contact: OECD Environment Directorate, Environment, Health and Safety Division 2 rue André-Pascal Paris Cedex 16 France Fax: (33-1) ehscont@oecd.org 16

17 FOREWORD This document is the Retrospective Performance Assessment (RPA) of the Test Guideline (TG) 488 on Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays. It has been developed to ensure that the Test Guideline 488 satisfies current OECD validation criteria, as described in Guidance Document 34 on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. The information provided in the RPA is based on the Detailed Review Paper (DRP) on Transgenic Rodent (TGR) Gene Mutation Assays, published in 2009 in the Series on Testing and Assessment (No. 103) and on unpublished supplementary analyses. It is expected that when the supplementary analyses are made publicly available, an annex including these supplementary analyses will be sent to the Joint Meeting for declassification and the current RPA will be updated to include this annex. The document was prepared by Canada and submitted to the Working Group of the National Coordinators for the Test Guidelines Progamme (WNT) for comments in October 2010, together with the draft TG on Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays. Comments were addressed and the WNT endorsed the RPA in April The Joint Meeting of the Chemicals Committee and Working Party on Chemicals, Pesticides and Biotechnology (Joint Meeting) agreed to the declassification of this document on 5 August This document is published under the responsibility of the Joint Meeting. 17

18 TRANSGENIC RODENT SOMATIC AND GERM CELL GENE MUTATION ASSAYS: RETROSPECTIVE PERFORMANCE ASSESSMENT Prepared for the Organisation for Economic Cooperation and Development (OECD) by George R. Douglas Mechanistic Studies Division Environmental Health Sciences and Research Bureau Healthy Environments and Consumer Safety Branch Health Canada OTTAWA, ONTARIO, CANADA, K1A 0K9 18

19 INTRODUCTION 1. The Detailed Review Paper (DRP) on Transgenic Rodent (TGR) Gene Mutation Assays, which has been approved and published by OECD (OECD, 2009), provides significant information and analysis pertinent to this review of the state of existing validation. This report also follows closely the validation criteria established by the OECD (OECD, 2005), which are used as the headings in the following text. 1) The rationale for the test method. This should include a clear statement of the scientific basis, regulatory purpose and need for the test. 2. Scientific basis. The scientific basis for TGR mutation assays has been described exhaustively in the DRP on TGR Assays. Briefly, TGR mutation assays are well-established assays that employ transgenic rats and mice which contain multiple copies of chromosomally integrated plasmid or phage DNA that harbour reporter genes for the detection of mutation. Mutations arising in a rodent are scored by recovering the vector and analysing the phenotype of the reporter gene in a bacterial host. TGR gene mutation assays allow mutations induced in a genetically neutral transgene (Cosentino and Heddle, 1996; 1999; Swiger et al., 2001) to be scored in any tissue of the rodent, and therefore circumvent many of the existing limitations associated with the study of in vivo gene mutation. TGR models for which sufficient data are available to permit evaluation include Muta tm mouse, Big Blue mouse and rat, lacz plasmid mouse, and gpt delta mouse and rat. Mutagenesis in the TGR models is normally assessed as a mutant frequency; if required, however, molecular analysis (i.e. DNA sequencing) can provide additional information on the exact nature of the mutations detected. The assays provide a unique opportunity to determine the ability of test agents to induce gene mutations in vivo in any tissue. Thus, TGR assays provide quick and reliable methods for detecting and quantifying gene mutations in vivo. They are useful for determining whether or not in vitro assays have produced false positive results, and for providing a sensitive and statistically reliable confirmation of marginal results in cancer bioassays of potentially genotoxic carcinogens. 3. Regulatory purpose and need for the test. The TGR test detects gene mutations in virtually any rodent tissue from which DNA can be obtained. While OECD Test Guidelines exist for a range of in vitro mutation assays that are capable of detecting chromosomal alterations or gene mutations, in vivo assays are required components of many genetic toxicity testing strategies. 4. For somatic cells in vivo, OECD Test Guidelines are currently available for assays capable of assessing induced chromosomal damage (TG 474 and 475). Yet, for gene mutations in vivo, there are no OECD Test Guidelines, aside from the little-used (Wahnschaffe et al., 2005) rodent Spot Test (TG 484). While there are some non-transgenic assays that can be used for analysis of gene mutations in vivo, none of these assays have an OECD Test Guideline, and they are limited by factors such as: the number of tissues in which genotoxicity can be measured; the state of understanding of the endpoint; the nature of the chemicals that can be detected; and cost. The TGR assay is the furthest advanced and most well characterized of the in vivo tests for gene mutation. 5. The OECD Working Group of National Coordinators of the Test Guidelines Program (WNT) has accepted the DRP (OECD, 2009) that describes and analyzes TGR mutation assays, and it has accepted the proposal to proceed to the development of a Test Guideline, subject to demonstration of sufficient validation. 19

20 6. TGR assays can fulfill three different regulatory needs or strategies. 1) TGR assays could be used as the primary in vivo test system for confirming positive in vitro gene mutation results. This strategy would have immediate application in some regulatory jurisdictions (e.g. Canadian Environmental Protection Act, New Substances Notification regulations) where it can be integrated immediately into existing regulatory frameworks. However, other regulatory testing jurisdictions may need to revise their testing requirements to accommodate this approach. 2) A more easily implemented approach would be to use TGR assays as the second in vivo test when the primary test [normally the in vivo micronucleus test (TG 474) or chromosomal aberration assay (TG 475)] is negative and the substance is positive in vitro. 3) TGR assays also can be used in case of tumour findings to determine if carcinogenicity in the target tissue is due to genotoxic or nongenotoxic mechanisms, knowing that further understanding of the mode of action impacts risk assessment approaches, and to confirm marginal responses with an assay of much greater statistical power and reproducibility. Furthermore, TGR studies can be performed simultaneously with TG 474 on the same animals, thus minimizing the use of animals and reducing the cost of the studies. 2) The relationship between endpoint(s) and the in vivo biological effect, and the toxicity of interest should be described. This should include a reference to scientific relevance of the effect(s) measured by the test method in terms of their mechanistic (biological) or empirical (correlative) relationship to the specific type of effect/toxicity of interest. Although the relationship may be mechanistic or correlative, test methods with biological relevance to the effect/toxicity being evaluated are preferred. 7. The endpoint of the TGR assays is in vivo gene mutation. Transgenes serve as surrogates for the genes that are targets for diseases resulting from mutation. Mutation cause both somatic diseases (Erikson, 2003) and heritable genetic diseases, and is the primary biological phenomenon of interest in the induction of cancer (Cleaver, 1969). The DRP contains an exhaustive analysis of the predictive value of the assay for the detection of mutations. This analysis showed that over 96% of the almost 33,000 mutant phenotypes detected in TGR assays are true genotypic mutations by DNA sequence analysis, thus, confirming and validating the exceptional positive predictivity of the assay for the mutation endpoint. 3) A formal detailed protocol for the test method should be readily available in the public domain. The protocol should be sufficiently detailed and should include, e.g., a description of the materials needed, such as specific cell types or construct or animal species that could be used for the test (if applicable), a description of what is measured and how it is measured, a description of how data will be analysed, decision criteria for evaluation of data and what are the criteria for acceptable test performance. 8. Details on the assay principles and the description of the multi-step experimental procedures are described in the literature and have been summarized in the DRP. A detailed internationallyharmonized protocol has been developed and published by the International Workshops on Genotoxicity Testing (IWGT; Heddle et al., 2000; Thybaud et al., 2003). Recommendations were made regarding the proper conduct of the TGR assay, which relate to treatment protocols, accepted characteristics, and post-treatment sampling procedures. Of particular importance in optimizing TGR protocols are two experimental variables, the administration time and the sampling time. Based on observations that mutations accumulate with each consecutive treatment, a repeated-dose regimen for a period of 28 days is recommended, with sampling at 3 days following the final treatment. If slowly proliferating tissues are of particular importance, then a longer sampling time of 28 days may be more appropriate. This protocol forms the basis for the TGR OECD Test Guideline. 20

21 4) The intra-test variability, repeatability, and reproducibility of the test method within and amongst laboratories should be demonstrated. Data should be available revealing the level of reproducibility and variability within and among laboratories over time. The degree to which biological variability affects the test method reproducibility should be addressed. 9. As of December 2007, according to the data that were available, 228 chemicals had been tested in TGR assays. While the identity of these chemicals included virtually all the common mutagens and a number of nonmutagens, the majority of these exposures did not use the newer IWGT protocol, with the majority of exposures being for 1-5 days with varying sampling times (OECD, 2009; Table 1; Table 4.6). Nevertheless, from the data-based mechanistic considerations underlying the IWGT recommendations it is highly unlikely that any positive outcomes derived from a non-iwgt protocol would be negative using the IWGT protocol. In contrast, since mutations accumulate with repeated treatments (because the reporter genes are neutral resulting in no selection for or against a mutant), it is possible that non-iwgt protocols with shorter treatment times could yield false negative outcomes for weak mutagens. The 28 day treatment time proposed in the IWGT protocol overcomes this hypothetical possibility. For example, as described in the DRP, two weak mutagens, acrylamide (Thybaud et al., 2003) and ethyl carbamate (urethane; Singer, unpublished), have been identified correctly using the IWGT protocol, confirming the advantage of this protocol. 10. The fact that the assay can be performed effectively over a wide variety of treatment and sampling times attests to its adaptability and plasticity. Accordingly, studies on inter- and intra-laboratory variation, and the sources of variability that were conducted prior to the establishment of the IWGT protocol (described below) can be considered as representative of the assay s performance, since these studies were conducted on chemicals that were known to be positive under the protocol conditions studied. 11. Furthermore, since the DRP data cut-off date of December 2007, there have been at least 23 additional studies (data from additional studies exist but were not available for this analysis) performed on standard mutagens and molecules with unknown genotoxic potential that attest to the performance of the IWGT protocols, i.e and (Table 2). These data are important because many of these studies, i.e. those performed by Contract Research Organizations (CROs) were conducted at the request of regulatory authorities, and/or were submitted to regulatory authorities, thereby demonstrating their existing routine regulatory use. Of particular significance in this regard are data on ethylmethanesulphonate (Gocke et al., 2009) that were used as the basis for a regulatory decision in the EU (Müller and Singer, 2009). Intralaboratory variability as a measure of repeatability. 12. Sources of variability in the experimental protocol that can affect the performance of the assay have been examined (Piegorsch et al., 1994, 1997). Such sources of variability include plate-toplate (within package), package-to-package (within animal) and animal-to-animal variability. Data from five laboratories were evaluated in detail. The results suggested that only scattered patterns of excess variability below the animal-to-animal level occur, but that, generally, excess variability is observed at the animal-to-animal level (Piegorsch et al., 1997). Statistical tests that may be used to reduce variability have been suggested (Carr and Gorelick, 1994, 1995; Piegorsch et al., 1995) and are components of the IWGT-recommended test protocol described by Thybaud et al. (2003). 21

22 13. Fung et al. (1998) studied the sources of variation from a tightly controlled experimental design. Sources of variation, including plates (within packaging reactions), packaging reactions (within animal) and between animals, were evaluated for extrabinomial variation. Although hardly any evidence of overdispersion was detected at the plate level, limited evidence of extra-binomial variation was observed at the packaging reaction level. There was, nevertheless, much stronger evidence of overdispersion at the animal level. Statistical tests for increasing trend in mutant frequency with increasing dose were also performed at the animal level. A significant doserelationship following exposure to N-nitrosodibenzylamine was detected by trend analysis in liver but not in bone marrow. A logistical model was used to further describe the dose-response relationship observed in N-nitrosodibenzylamine-treated liver tissue. 14. The DRP compared results for several chemicals in Muta mouse liver and bone marrow and in Big Blue mouse liver. These results are obtained from querying the DRP database (TRAID) and are not the result of any collaborative study. Muta mouse liver and bone marrow and Big Blue mouse liver are the only combinations where significant experimental data are currently available to allow comparisons among studies. The majority of the chemicals examined are strong mutagens and produce, as expected, positive results in the TGR assays. Among these are only a small number of instances in which ENU returned inconsistent results when inadequate sampling times were used. Nevertheless, overall, the data in the DRP strongly indicate that similar qualitative results are obtained in different studies and that the results are reproducible. 15. A nonstatistical comparison (Table 2) of intralaboratory reproducibility of data from studies using the IWGT protocols shows a qualitatively reproducible response from multiple experiments showing the mutagenicity of the weak mutagen ethyl carbamate (urethane). 16. A statistical analysis of old and IWGT protocols was performed on data from a Muta tm mouse study carried out using, ethyl carbamate, a weak mutagen (Douglas and Williams, unpublished). A weak mutagen was chosen for analysis in order to provide a rigorous test of the robustness of the protocols in different tissues. The analysis showed that while the IWGT protocols yielded similar (positive) results in both rapidly (bone marrow) and slowly dividing (liver) tissue, the old protocols did not produce positive results in a slowly dividing tissue (liver). Similar results were found previously for acrylamide, another weak mutagen (Thybaud et al., 2003). The lack of a positive response in slowly dividing tissue is likely the result of insufficient accumulation of (weak) mutagenic DNA lesions, and their fixation as mutations through cell division when using a short administration time. In contrast, the longer 28 day administration time in the IWGT protocols provide sufficient accumulation and fixation of mutations in both slowly and rapidly dividing tissues, and result in mutant frequencies that are at least as high if not higher than after 7 days of dosing. 17. Nevertheless, in addition to demonstrating the robustness of the IWGT protocols, these results show that data produced with older protocols that used shorter administration times (OECD, 2009) are still relevant to the consideration of overall TGR assay performance if performed on strong mutagens, or when positive results are reported. 22

23 Interlaboratory variability as a measure of reproducibility 18. A collaborative study involving 26 laboratories examined ENU mutagenesis in eight organs of Muta mouse liver, spleen, bone marrow, brain, lung, kidney, urinary bladder and heart following a single intraperitoneal injection of 150 mg ENU/kg bw (Collaborative Study Group for the Transgenic Mouse Mutation Assay, 1996). Many of the laboratories involved in this study had no prior experience using TGR assays, and the study was preceded by a 1-day training session for all participants. A standard DNA sample was analysed by all laboratories; the results from only two of the laboratories varied over two-fold from the mean mutant frequency, and there was an overall high level of concordance in the mutant frequencies obtained using this standard sample. Among the organs tested, similar conclusions were reached by most laboratories regarding whether a positive or negative result was obtained. As expected, the potent mutagen ENU increased the mutant frequency in all organs except brain. However, the study design did not allow a rigorous statistical evaluation of the data or the extent of interlaboratory variation (Collaborative Study Group for the Transgenic Mouse Mutation Assay, 1996). 19. The mutagenicity of DMN was evaluated in three laboratories using common liver samples from Muta mouse and Big Blue mice. The liver samples compared were obtained from mice treated once with either saline or DMN (10 mg/ml, 14-day sampling time). Each assay gave an increased mutant frequency for the DMN-treated livers when compared with the saline control mutant frequencies (Tinwell et al., 1995). 20. A collaborative study examined mouse germ cell mutagenesis of ENU, isopropylmethanesulphonate (ipms) and methylmethanesulphonate (MMS) in both Muta mouse and Big Blue mice. Both testicular DNA and epididymal sperm DNA were evaluated, and a range of sampling times, from 3 to 100 days, was examined. ENU and ipms were found to be mutagenic to both testicular DNA and epididymal sperm DNA. MMS was not mutagenic under any test condition. The authors concluded that a good level of qualitative agreement was obtained for the two assays and for the same assays conducted in different laboratories (Ashby, 1995; Ashby et al., 1997). In addition, the comparability of the gpt delta assay with these two assays has also been established (Swiger et al., 2001). 21. With respect to recent studies employing the IWGT protocol (28 + 3) a Japanese interlaboratory study on the performance of the gpt delta rat assay for detecting the mutagenicity of 2,4- diaminotoluene, 2,6-diaminotoluene, and aristolochic acid has recently been completed. The study confirms the interlaboratory comparability of this protocol (Table 2; Douglas and Williams, unpublished). 22. In summary, the results of a number studies carried out using a variety of experimental protocols with a number of mutagens suggest that the TGR assays show good qualitative reproducibility in both somatic and germ cells and quantitative reproducibility over a range of conditions covering protocols employing shorter exposure conditions, as well as studies using the IWGT protocol across different laboratories. 5) The test method's performance must be demonstrated using a series of reference chemicals preferably coded to exclude bias. A sufficient number of the reference chemicals should have been tested under code to exclude bias. 23. The DRP (OECD, 2009) shows that a full range of representative and reference agents have been tested under a variety of experimental conditions in TGR assays. Analysis of predictive statistics in the DRP showed that TGR assays perform similarly to, or better than, other assays for gene 23

24 mutation; for example, the positive and negative cancer predictivities for the TGR and Salmonella assays were almost identical. Furthermore, Table 2 demonstrates a range of reference and representative agents tested recently using the IWGT protocol, including many tested by contract research organizations for regulatory purposes. 24. Guidance Document 34 (OECD, 2005) states that it is preferable, but it is not a requirement, that agents be coded so that their identity is not known during the testing and data collection, in order to avoid bias in the results due to prior knowledge of the expected responses. While most of the data available are not from coded samples, there is little danger of bias because the methodology is mostly analytical (i.e. not subjective), and there is adequate duplication across laboratories among the chemicals tested, which provides a check against the introduction of bias. 6) The performance of the test method should have been evaluated in relation to relevant toxicity data as well as information from the relevant target of concern. In the case of a substitute test method adequate data should be available to permit a reliable analysis of the performance and comparability of the proposed substitute test method with that of the test it is designed to replace. 25. The TGR assay is not a substitute assay; it is a new definitive test or adjunct test method (depending on the use context) that is designed to fill a gap in the in vivo mutation assays that are available for regulatory testing. As described above in (section 2), it faithfully detects the endpoint that it is designed to detect. 7) Normally, all data supporting the validity of a test method should have been obtained in accordance with the principles of GLP. Aspects of data collection not performed according to GLP should be clearly identified and their potential impact on the validation status of the test method should be indicated. 26. Much of the data in Table 2 were derived from full GLP studies, or studies conducted in the spirit of GLP. However, it should be recognized that because this assay is not a rapid screening test, and is experimentally complex, the experiments require a significant planning effort which results in a high level of procedural and formal quality even when not conducted under full GLP. In the foreseeable use for regulatory purposes, GLP compliance will be an obvious prerequisite, and many studies have been conducted in that way already. 8) All data supporting the assessment of the validity of the test method should be available for expert review. The detailed test method protocol should be readily available and in the public domain. The data supporting the validity of the test method should be organised and easily accessible to allow for independent review(s), as appropriate. The test method description should be sufficiently detailed to permit an independent laboratory to follow the procedures and generate equivalent data. Benchmarks should be available by which an independent laboratory can itself assess its proper adherence to the protocol. 27. The detailed IWGT test method protocol is readily available and in the public domain (Heddle et al., 2000; Thybaud et al., 2003). The test method description is sufficiently detailed to permit an independent laboratory to follow the procedures and generate equivalent data (see Table 2 and Supplementary Analysis). The recommended protocol contains sufficient benchmark indicators by which a laboratory can assess its proper adherence to the protocol and the quality of the results obtained. The DRP (OECD, 2009) contains well-organized, extensive data supporting the overall validity of the test as performed prior to the IWGT protocol modifications. Table 2 contains additional summary information demonstrating the routine use of the new IWGT protocol, some of 24

25 which cannot be released in more detail because the data are unpublished or proprietary. The Supplementary Analysis contains more detailed data for some of the studies in Table 2, but which are available on a restricted basis until published. CONCLUSION 28. Based on the above summary, it is concluded that there is sufficient evidence of validation of the TGR mutation assay to support the establishment of a Test Guideline. ACKNOWLEDGEMENTS I thank the many experts who provided comments and suggestions during the preparation of this document (especially Michael Cimino, Robert Heflich, Hans Joerg Martus, Kenichi Masumura, Takehiko Nohmi, Tim Singer,Véronique Thybaud, and John Heddle). I am also grateful to Tim Singer, Takehiko Nohmi, Kenichi Masumura, Ed Riccio, and Mark Ballentyne for providing unpublished data. 25

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