The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Aix Marseille University CEA CNRS Institute of Biosciences and Biotechnologies of Aix Marseille Saint Paul Lez Durance France

Anses French Agency for Food Environmental and Occupational Health and Safety Fougeres Laboratory Toxicology of Contaminants Unit Fougères France

Biocev 1st Medical Faculty Charles University Vestec Czech Republic

Biology and Environmental Toxicology Group Faculty of Science Universidad Nacional de Educación a Distancia Madrid Spain

Biomedical Center Faculty of Medicine in Pilsen Charles University Pilsen Czech Republic

Biomedical Sciences Department University of Bradford Bradford UK

Centre for Environmental and Marine Studies and Department of Biology University of Aveiro Campus Universitário de Santiago Aveiro Portugal

Centre for Environmental Radioactivity Oslo Norway

Centre for Environmental Sciences Hasselt University Diepenbeek Belgium

Centre for Radiobiology and Biological Dosimetry Institute of Nuclear Chemistry and Technology Warsaw Poland

Centre of BioMedical Research SGPGIMS Campus Lucknow India

Department Circular Economy and Environment the Netherlands Organisation for Applied Scientific Research TNO Utrecht The Netherlands

Department NEUROFARBA Section Pharmacology and Toxicology University of Florence Florence Italy

Department of Biological Engineering Massachusetts Institute of Technology Cambridge MA USA

Department of Biology CBMA Centre of Molecular and Environmental Biology University of Minho Braga Portugal

Department of Biology University of Rzeszow Rzeszow Poland

Department of Biotechnology University of Rzeszow Rzeszow Poland

Department of Cardiothoracic Surgery University of the Free State Bloemfontein South Africa

Department of Chemical Engineering Massachusetts Institute of Technology Cambridge MA USA

Department of Environmental Biological and Pharmaceutical Sciences and Technologies University of Campania Luigi Vanvitelli Caserta Italy

Department of Food Environmental and Nutritional Sciences Università degli Studi di Milano Milan Italy

Department of Genetic Toxicology and Cancer Biology National Institute of Biology Ljubljana Slovenia

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Department of Genomic Medicine and Environmental Toxicology Instituto de Investigaciones Biomédicas Universidad Nacional Autónoma de México CU Mexico City Mexico

Department of Human Sciences and Quality of Life Promotion San Raffaele University Rome Italy

Department of Molecular Biology and Translational Research Institute of Rural Health Lublin Poland

Department of Molecular Biology of Cancer Institute of Experimental Medicine of the Czech Academy of Sciences Prague Czech Republic

Department of Nutrition University of Oslo Oslo Norway

Department of Pharmaceutical Sciences Unit of Public Health University of Perugia Perugia Italy

Department of Pharmacology and Toxicology School for Nutrition and Translational Research in Metabolism Maastricht University Maastricht The Netherlands

Department of Pharmacology and Toxicology University of Navarra Pamplona Spain

Department of Public Health Section of Environmental Health University of Copenhagen Copenhagen Denmark

Division of Climate and Environmental Health Norwegian Institute of Public Health Oslo Norway

Environmental Health Department National Institute of Health Porto Portugal

EPIUnit Instituto de Saúde Pública Universidade do Porto Porto Portugal

FARMOVS Bloemfontein South Africa

Food and Drug Administration National Center for Toxicological Research Division of Genetic and Molecular Toxicology Jefferson AR USA

Genetics and Biotechnology Department and Veterinary and Animal Research Centre Universidade de Trás os Montes e Alto Douro Vila Real Portugal

Global Product Stewardship Human Safety The Procter and Gamble Co Cincinnati OH USA

H and TRC Health and Technology Research Center ESTeSL Escola Superior de Tecnologia da Saúde Instituto Politécnico de Lisboa Lisbon Portugal

Health Effects Laboratory Department of Environmental Chemistry NILU Norwegian Institute for Air Research Kjeller Norway

Helix3 Inc RTP Morrisville NC USA

Henkel AG and Co KGaA Düsseldorf Germany

IdiSNA Navarra Institute for Health Research Pamplona Spain

Institute of Cancer Research Internal Medicine 1 Medical University Vienna Vienna Austria

Institute of Epigenetics and Stem Cells Helmholtz Zentrum München Munich Germany

Institute of Pharmacology and Toxicology University of Würzburg Würzburg Germany

Instituto de Investigación Biomédica de A Coruña A Coruña Spain

Laboratoire Ecologie fonctionnelle et Environnement Université de Toulouse CNRS INPT UPS Toulouse France

Laboratory for Integrative and Translational Research in Population Health Porto Portugal

Laboratory of Ecotoxicology Faculty of Natural Sciences and Museum National University of La Plata La Plata Argentina

Mutagenesis Unit Institute for Medical Research and Occupational Health Zagreb Croatia

Nanomaterial Toxicology Group CSIR Indian Institute of Toxicology Research Lucknow India

NorGenoTech AS Oslo Norway

Norwegian Scientific Committee for Food and Environment Oslo Norway

NOVA National School of Public Health Public Health Research Centre Universidade NOVA de Lisboa Lisbon Portugal

Oxidative Stress Group Department of Cell Biology Microbiology and Molecular Biology University of South Florida Tampa FL USA

Oxidative Stress Group Department of Environmental Health Sciences Florida International University Miami FL USA

School of Biological and Marine Sciences University of Plymouth Plymouth UK

School of Chemistry and Bioscience Faculty of Life Sciences Bradford University Bradford UK

School of Pharmacy and Life Sciences The Robert Gordon University Aberdeen Scotland

Toxalim Université de Toulouse INRAE ENVT INP Purpan UPS Toulouse France

Toxicology Department AC MARCA Group L'Hospitalet de Llobregat Barcelona Spain

Translational Biomedicine Laboratory Graduate Program of Health Sciences University of Southern Santa Catarina Criciuma Brazil

Triskelion BV Zeist The Netherlands

Unit of Clinical and Molecular Epidemiology IRCCS San Raffaele Roma Rome Italy

Universidade da Coruña Grupo DICOMOSA CICA Centro Interdisciplinar de Química e Bioloxía Departamento de Psicología Facultad de Ciencias de la Educación A Coruña Spain

Universidade da Coruña Grupo NanoToxGen CICA Centro Interdisciplinar de Química e Bioloxía Departamento de Biología Facultad de Ciencias A Coruña Spain

VITO Health VITO Mol Belgium

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DNA damage in oral mucosal epithelial cells cultured in complex and xenobiotic-free media: a comparison study

Mechanisms underlying low mutation rates in mammalian oocytes and preimplantation embryos

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project