Mutagenicity Testing: Regulatory Guidelines and Current Needs

Chemicals interact with genes, resulting in harmful mutations, potentially causing cancer and damaging our offspring, and this is a major environmental concern today. Considerable attention has been given by regulatory bodies across the globe to develop objective methods for utilizing mutagenicity information in regulatory decision-making. Due to industrialization and advances in technology, a number of new drugs, cosmetics, pesticides, food additives, and other industrial chemicals are released into the market worldwide. Various in vitro and in vivo methods are followed for screening of mutagenicity potential before being accepted by FDA, Environmental Protection Agency (EPA), and other regional regulatory bodies. Ames test is one of the best examples for testing mutagenicity, while some new methods are still at the stage of validation. Mutagenicity methods have different levels of sensitivity and specificity, which contributes to false positive and false negative results. As no one method yields full proof confirmation for mutagenicity, a tiered approach using two or more tests is generally suggested for proper evaluation of a product. Regulatory bodies of various countries have designed framework for mutagenicity testing to protect humans, other living beings, and environment at large. Despite the best efforts, loopholes between mutagenicity evaluation outcome and actual carcinogenicity occurrence still exist. This can be addressed only by improving the current testing strategy and development of new mutagenicity testing assays.

[1]  Neal F. Cariello,et al.  Comparison of the computer programs DEREK and TOPKAT to predict bacterial mutagenicity. Deductive Estimate of Risk from Existing Knowledge. Toxicity Prediction by Komputer Assisted Technology. , 2002, Mutagenesis.

[2]  Y. Katsuura,et al.  Development of an in vivo gene mutation assay using the endogenous Pig‐A gene: I. Flow cytometric detection of CD59‐negative peripheral red blood cells and CD48‐negative spleen T‐cells from the rat , 2008, Environmental and molecular mutagenesis.

[3]  Michael D Waters,et al.  Approaches for identifying germ cell mutagens: Report of the 2013 IWGT workshop on germ cell assays(☆). , 2015, Mutation research. Genetic toxicology and environmental mutagenesis.

[4]  Awadhesh N Jha,et al.  Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. , 2006, Mutation research.

[5]  Zbigniew Darzynkiewicz,et al.  Cytometric assessment of DNA damage in relation to cell cycle phase and apoptosis , 2005, Cell proliferation.

[6]  Takanori Nakamura,et al.  Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity? , 2010, Journal of nucleic acids.

[7]  Teizo Fujita,et al.  Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria , 1993, Cell.

[8]  Silvio Albertini,et al.  Evaluation of the GreenScreen GADD45alpha-GFP indicator assay with non-proprietary and proprietary compounds. , 2009, Mutation research.

[9]  T Sofuni,et al.  Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards. , 2007, Mutation research.

[10]  J. T. Macgregor,et al.  The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program. , 1983, Mutation research.

[11]  Raymond R Tice,et al.  Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. , 2007, Mutation research.

[12]  Julie Clements,et al.  Mouse lymphoma thymidine kinase gene mutation assay: Follow‐up International Workshop on Genotoxicity Test Procedures, New Orleans, Louisiana, April 2000 , 2002, Environmental and molecular mutagenesis.

[13]  G. Cross,et al.  Glycolipid anchoring of plasma membrane proteins. , 1990, Annual review of cell biology.

[14]  Takashi Omori,et al.  Statistical issues in the use of the comet assay. , 2008, Mutagenesis.

[15]  S. Das,et al.  ortho-Substituent effects on the in vitro and in vivo genotoxicity of benzidine derivatives. , 1993, Mutation research.

[16]  M. G. Low.  The glycosyl-phosphatidylinositol anchor of membrane proteins. , 1989, Biochimica et biophysica acta.

[17]  M. Hollstein,et al.  Knock-in mice with a chimeric human/murine p53 gene develop normally and show wild-type p53 responses to DNA damaging agents: a new biomedical research tool , 2001, Oncogene.

[18]  M. Kirsch‐Volders,et al.  The in vitro MN assay in 2011: origin and fate, biological significance, protocols, high throughput methodologies and toxicological relevance , 2011, Archives of Toxicology.

[19]  Isao Yoshimura,et al.  Mouse lymphoma thymidine kinase gene mutation assay: Follow‐up meeting of the international workshop on Genotoxicity testing—Aberdeen, Scotland, 2003—Assay acceptance criteria, positive controls, and data evaluation , 2006, Environmental and molecular mutagenesis.

[20]  Martha M Moore,et al.  Use of genetic toxicology information for risk assessment , 2005, Environmental and molecular mutagenesis.

[21]  E. Zeiger,et al.  Quantitative approaches for assessing dose–response relationships in genetic toxicology studies , 2013, Environmental and molecular mutagenesis.

[22]  T. Kinoshita,et al.  Paroxysmal nocturnal hemoglobinuria: An acquired genetic disease , 1999, American journal of hematology.

[23]  D. Smart.  Genotoxicity of topoisomerase II inhibitors: an anti-infective perspective. , 2008, Toxicology.

[24]  Paul Cahill,et al.  GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests. , 2010, Mutation research.

[25]  Richard M Walmsley,et al.  Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay. , 2008, Mutagenesis.

[26]  Ming K. Lee,et al.  Aflatoxin‐B exposure does not lead to p53 mutations but results in enhanced liver cancer of Hupki (human p53 knock‐in) mice , 2006, International journal of cancer.

[27]  R. Brodsky,et al.  PIG-A mutations in paroxysmal nocturnal hemoglobinuria and in normal hematopoiesis , 2006, Leukemia & lymphoma.

[28]  M. Shelby,et al.  Micronuclei as an index of cytogenetic damage: Past, present, and future , 1991, Environmental and molecular mutagenesis.

[29]  S. Boulton,et al.  Playing the end game: DNA double-strand break repair pathway choice. , 2012, Molecular cell.

[30]  S. De Flora,et al.  Mutagenesis and cardiovascular diseases Molecular mechanisms, risk factors, and protective factors. , 2007, Mutation research.

[31]  J. Harvey,et al.  Genotoxicity screening via the γH2AX by flow assay. , 2011, Mutation research.

[32]  Isao Yoshimura,et al.  Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002. , 2003, Mutation research.

[33]  Richard M Walmsley,et al.  Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP 'GreenScreen HC' genotoxicity assay. , 2009, Mutagenesis.

[34]  Serena Nik-Zainal,et al.  Mechanisms underlying mutational signatures in human cancers , 2014, Nature Reviews Genetics.

[35]  J. Kleinjans,et al.  Performance of in vitro γH2AX assay in HepG2 cells to predict in vivo genotoxicity. , 2012, Mutagenesis.

[36]  I. Lambert,et al.  Detailed review of transgenic rodent mutation assays. , 2005, Mutation research.

[37]  H. van Steeg,et al.  Mechanisms of non-genotoxic carcinogens and importance of a weight of evidence approach. , 2009, Mutation research.

[38]  X. Castells,et al.  Modelling mutational landscapes of human cancers in vitro , 2014, Scientific Reports.

[39]  Yves Pommier,et al.  γH2AX and cancer , 2008, Nature Reviews Cancer.

[40]  Annick Lauwers,et al.  Evaluation of the Vitotox and RadarScreen assays for the rapid assessment of genotoxicity in the early research phase of drug development. , 2009, Mutation research.

[41]  Richard M Walmsley,et al.  High-specificity and high-sensitivity genotoxicity assessment in a human cell line: validation of the GreenScreen HC GADD45a-GFP genotoxicity assay. , 2006, Mutation research.

[42]  J. Holme,et al.  Genotoxicity studies with paracetamol. , 1984, Mutation research.

[43]  Andreas Czich,et al.  Follow‐up actions from positive results of in vitro genetic toxicity testing , 2011, Environmental and molecular mutagenesis.

[44]  N. Kruhlak,et al.  An analysis of genetic toxicity, reproductive and developmental toxicity, and carcinogenicity data: I. Identification of carcinogens using surrogate endpoints. , 2006, Regulatory toxicology and pharmacology : RTP.

[45]  Romualdo Benigni,et al.  Structure alerts for carcinogenicity, and the Salmonella assay system: a novel insight through the chemical relational databases technology. , 2008, Mutation research.

[46]  B. S. Kim,et al.  Statistical analysis of in vivo rodent micronucleus assay. , 2000, Mutation research.

[47]  David T. W. Jones,et al.  Signatures of mutational processes in human cancer , 2013, Nature.

[48]  W. Muster,et al.  Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup report , 2000, Environmental and molecular mutagenesis.

[49]  T. Skopek,et al.  In vivo transgenic mutation assays , 2000, Environmental and molecular mutagenesis.

[50]  A. Gennery,et al.  Primary immunodeficiencies associated with DNA-repair disorders , 2010, Expert Reviews in Molecular Medicine.

[51]  E Zeiger,et al.  Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing. , 2007, Mutation research.

[52]  J. Kleinjans,et al.  Application of toxicogenomics to study mechanisms of genotoxicity and carcinogenicity. , 2009, Toxicology letters.

[53]  W. Schoonen,et al.  The development of RAD51C, Cystatin A, p53 and Nrf2 luciferase-reporter assays in metabolically competent HepG2 cells for the assessment of mechanism-based genotoxicity and of oxidative stress in the early research phase of drug development. , 2010, Mutation research.

[54]  David Lovell,et al.  Compilation and use of genetic toxicity historical control data. , 2011, Mutation research.

[55]  S. Frank.  Somatic evolutionary genomics: Mutations during development cause highly variable genetic mosaicism with risk of cancer and neurodegeneration , 2010, Proceedings of the National Academy of Sciences.

[56]  R. Walmsley,et al.  Interlaboratory assessment of the GreenScreen HC GADD45a-GFP genotoxicity screening assay: an enabling study for independent validation as an alternative method. , 2008, Mutation research.

[57]  Thomas Steger-Hartmann,et al.  Computational prediction of the chromosome-damaging potential of chemicals. , 2006, Chemical research in toxicology.

[58]  N. Kruhlak,et al.  In silico screening of chemicals for bacterial mutagenicity using electrotopological E-state indices and MDL QSAR software. , 2005, Regulatory toxicology and pharmacology : RTP.

[59]  Lutz Müller,et al.  Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens I. Sensitivity, specificity and relative predictivity. , 2005, Mutation research.

[60]  Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock‐in (Hupki) mouse model , 2010 .

[61]  Richard M Walmsley,et al.  Development and Validation of a Higher Throughput Screening Approach to Genotoxicity Testing Using the GADD45a-GFP GreenScreen HC Assay , 2009, Journal of biomolecular screening.

[62]  M. Stratton,et al.  Mutational signatures: the patterns of somatic mutations hidden in cancer genomes , 2014, Current opinion in genetics & development.

[63]  M. Olivier,et al.  Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid. , 2013, Mutation research.

[64]  Y. Endo,et al.  Molecular cloning of murine pig-a, a gene for GPI-anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and genetic locus. , 1994, Genomics.

[65]  E. Zeiger,et al.  Derivation of point of departure (PoD) estimates in genetic toxicology studies and their potential applications in risk assessment , 2014, Environmental and molecular mutagenesis.

[66]  R. Tice,et al.  Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing , 2000, Environmental and molecular mutagenesis.

[67]  David Kirkland,et al.  Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens III. Appropriate follow-up testing in vivo. , 2005, Mutation research.

[68]  Lutz Müller,et al.  Strategies in case of positive in vivo results in genotoxicity testing. , 2011, Mutation research.

[69]  G. Speit,et al.  The Comet Assay (Single-Cell Gel Test) , 1999 .