Section 4.5 Genotoxicity Second edition (2020) Hazard Identification and Characterization CONTENTS LIST OF ABBREVIATIONS 4-3 LIST OF CONTRIBUTORS 4-6 4.5 Genotoxicity 4-8 4.5.1 Introduction 4-8 Risk analysis context and problem formulation 4-10 Decision-tree for assessing the mutagenicity of substances that can be found in food 4-12 4.5.2 Tests for genotoxicity 4-19 Bacterial mutagenicity 4-23 In vitro mammalian cell mutagenicity 4-23 (a) Forward gene mutation tests using the Tk gene 4-24 (b) Forward gene mutation tests using the Hprt and Xprt genes 4-24 In vivo mammalian cell mutagenicity 4-25 (a) Somatic cell assays 4-25 (b) Germ cell assays 4-26 In vitro chromosomal damage assays 4-27 (a) Chromosomal aberration assay 4-27 (b) Micronucleus (MN) assay 4-28 (c) TK assay in mammalian cells 4-28 In vivo chromosomal damage assays 4-29 (a) Chromosomal aberration assay 4-29 (b) Micronucleus (MN) assay 4-29 In vitro DNA damage/repair assays 4-30 In vivo DNA damage/repair assays 4-30 This text updates section 4.5 of Chapter 4, Hazard Identification and Characterization: Toxicological and Human Studies, of Environmental Health Criteria 240 (EHC 240), which was originally published in 2009. It was developed through an expert consultation and further advanced following comments received through a public consultation in December 2019. For abbreviations used in the text, the reader may refer to the list of abbreviations at the front of this section. Definitions of select terms may be found in the glossary in Annex 1 of EHC 240 (http://www.inchem.org/ documents/ehc/ehc/ehc240_annex1.pdf). 4-1 EHC 240: Principles for Risk Assessment of Chemicals in Food (a) Comet (single-cell gel electrophoresis) assay 4-30 (b) DNA adduct assays 4-32 (c) Unscheduled DNA synthesis (UDS) assay in mammalian liver 4-32 4.5.3 Identification of relevant studies 4-33 4.5.4 Interpretation of test results 4-34 Presentation and categorization of results 4-35 (a) Assessing whether results of an assay are positive, negative or equivocal for genotoxicity 4-37 (b) Assessing data quality 4-38 Weighting and integration of results 4-46 Adequacy of the genotoxicity database 4-49 Mutagenic mode of action and adverse outcomes 4-50 Integration of carcinogenicity and mutagenicity 4-53 4.5.5 Approaches for evaluating data-poor substances 4-56 In silico approaches 4-56 (a) Available tools (QSARs, SARs/ structural alerts) for mutagenicity 4-57 (b) Confidence in approaches 4-57 (c) Mutagenicity assessment 4-63 Threshold of toxicological concern (TTC) 4-65 Grouping and read-across approaches 4-68 4.5.6 Considerations for specific compounds 4-71 Mixtures 4-71 Flavouring agents 4-73 Metabolites in crops/food-producing animals, degradation products and impurities 4-75 Secondary metabolites in enzyme preparations 4-79 4.5.7 Recent developments and future directions 4-81 Novel in vivo genotoxicity approaches 4-82 Novel in vitro genotoxicity approaches 4-82 Adverse outcome pathways for mutagenicity 4-90 Quantitative approaches for safety assessment 4-92 4.5.8 References 4-93 4-2 Hazard Identification and Characterization List of abbreviations ACD Advanced Chemistry Development, Inc. ADI acceptable daily intake AOP adverse outcome pathway ARfD acute reference dose ATSDR Agency for Toxic Substances and Disease Registry (USA) BMD benchmark dose CAS Chemical Abstracts Service CCRIS Chemical Carcinogenesis Research Information System CEBS Chemical Effects in Biological Systems Cefic European Chemical Industry Council CHL Chinese hamster lung CHO Chinese hamster ovary CTD Comparative Toxicogenomics Database DDI DNA damage–inducing DNA deoxyribonucleic acid ECHA European Chemicals Agency EFSA European Food Safety Authority EHC Environmental Health Criteria EU European Union EURL ECVAM European Union Reference Laboratory for alternatives to animal testing FAO Food and Agriculture Organization of the United Nations GENE-TOX Genetic Toxicology Data Bank GLP Good Laboratory Practice gpt glutamic–pyruvic transaminase HBGV health-based guidance value Hprt/HPRT hypoxanthine–guanine phosphoribosyl transferase HTRF Homogeneous Time-Resolved Fluorescence IATA Integrated Approaches to Testing and Assessment ICH International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use INCHEM Internationally Peer Reviewed Chemical Safety Information IPCS International Programme on Chemical Safety IRIS Integrated Risk Information System (USA) ISS Istituto Superiore di Sanità (Italy) 4-3 EHC 240: Principles for Risk Assessment of Chemicals in Food ISSMIC Istituto Superiore di Sanità database on in vivo mutagenicity (micronucleus test) ISSSTY Istituto Superiore di Sanità database on in vitro mutagenicity in Salmonella typhimurium (Ames test) JECDB Japanese Existing Chemical Data Base JECFA Joint FAO/WHO Expert Committee on Food Additives JMPR Joint FAO/WHO Meeting on Pesticide Residues LRI Long-range Research Initiative MAK maximum workplace concentration MN micronucleus/micronuclei MOA mode of action MOE margin of exposure NGS next-generation DNA sequencing NIHS National Institute of Health Sciences (Japan) NOAEL no-observed-adverse-effect level NOGEL no-observed-genotoxic-effect level NTP National Toxicology Program (USA) OECD Organisation for Economic Co-operation and Development PAH polycyclic aromatic hydrocarbon PCR polymerase chain reaction Pig-a phosphatidylinositol glycan complementation group A qPCR quantitative polymerase chain reaction QSAR quantitative structure–activity relationship REACH Registration, Evaluation, Authorisation and Restriction of Chemicals RNA ribonucleic acid RT-qPCR reverse transcription quantitative polymerase chain reaction S9 9000 × g supernatant fraction from rat liver homogenate SAR structure–activity relationship SciRAP Science in Risk Assessment and Policy SYRCLE Systematic Review Centre for Laboratory Animal Experimentation Td threshold dose TDI tolerable daily intake T.E.S.T. Toxicity Estimation Software Tool TG test guideline; thioguanine TIMES tissue metabolism simulator 4-4 Hazard Identification and Characterization Tk/TK thymidine kinase ToxRTool Toxicological data Reliability Assessment Tool TTC threshold of toxicological concern UDS unscheduled DNA synthesis USA United States of America USEPA United States Environmental Protection Agency USFDA United States Food and Drug Administration WHO World Health Organization WOE weight of evidence Xprt/XPRT xanthine–guanine phosphoribosyl transferase 4-5 EHC 240: Principles for Risk Assessment of Chemicals in Food List of contributors Dr Virunya Bhat PAHO/WHO Collaborating Centre on Food Safety, Water Quality and Indoor Environment, NSF International, Ann Arbor, Michigan, United States of America (USA) Emeritus Professor Alan R. Boobis (co-lead author) National Heart & Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom Dr Riccardo Crebelli Istituto Superiore di Sanità, Rome, Italy Dr Nathalie Delrue Test Guidelines Programme, Environment, Health and Safety Division, Environment Directorate, Organisation for Economic Co-operation and Development, Paris, France Professor David Eastmond (co-lead author) Department of Molecular, Cell, and Systems Biology, University of California, Riverside, California, USA Dr Susan Page Felter Mason, Ohio, USA Dr Rainer Guertler Federal Institute for Risk Assessment (BfR), Berlin, Germany Professor Andrea Hartwig Karlsruhe, Germany Dr Frank Le Curieux European Chemicals Agency, Helsinki, Finland Professor Angelo Moretto Department of Biomedical and Clinical Sciences, University of Milan, and International Centre for Pesticides and Health Risk Prevention, Luigi Sacco Hospital, Milan, Italy Professor Pasquale Mosesso Department of Ecological and Biological Sciences, Università degli Studi della Tuscia, Viterbo, Italy Dr Utz Mueller Australian Pesticides and Veterinary Medicines Authority, Kingston, Australian Capital Territory, Australia 4-6 Hazard Identification and Characterization Dr Takehiko Nohmi Biological Safety Research Center, National Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo, Japan Dr Grace Patlewicz National Center for Computational Toxicology, United States Environmental Protection Agency, Durham, North Carolina, USA Professor David H. Phillips King’s College London, London, United Kingdom Dr Andrea Richarz Institute for Health and Consumer Protection, Joint Research Centre, European Commission, Ispra, Italy Dr Raymond R. Tice National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina, USA Dr Paul A. White Genetic Toxicology Group, Environmental Health Sciences & Research Bureau, Environmental & Radiation Health Sciences Directorate, Healthy Environments & Consumer Safety Branch, Health Canada, Ottawa, Ontario, Canada Dr Kristine L. Witt National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina, USA 4-7 EHC 240: Principles for Risk Assessment of Chemicals in Food 4.5 Genotoxicity 4.5.1 Introduction The study of toxic effects on the inherited genetic material in cells originated with the experiments of Muller (1927), who observed “artificial transmutation of the gene” by ionizing radiation in the fruit fly, Drosophila melanogaster. Chemically induced mutation also has a long history, with the first scientific publication, using Muller’s fruit fly model, describing mutations arising from exposure to sulfur mustard (Auerbach, Robson & Carr, 1947). A key event stimulating the development and validation of genetic toxicity