A proposal for a novel rationale for critical effect size in dose-response analysis based on a multi-endpoint in vivo study with methyl methanesulfonate.

Methyl methanesulfonate, a well-known direct-acting genotoxicant, was assessed in a multi-endpoint study in rats using six closely spaced dose levels. The main goal of the study was to investigate the genotoxic response at very low doses and to analyse this response with dedicated statistical tools in order to find a Point of Departure (PoD) and related metrics. Software packages like PROAST or EPA-BMDS require the toxicologist to define a so-called critical effect size (CES) or benchmark response (BMR) and this choice has a large impact on the result of the PoD calculation. Currently, increases of 5%, 10% or 1 standard deviation over concurrent vehicle controls have been proposed for CES/BMR, values that may or may not be suited for all genotoxicity endpoints. Based on the data obtained in this study, we propose an endpoint specific CES approach that reflects the typical evaluation process of a regulatory acceptable genotoxicology study. However, we are aware that this ratio-based CES strategy will need to be more fully developed with additional experimentation and should be mainly seen as a starting point for scientific discussion.

[1]  J. T. Macgregor,et al.  International Pig‐a gene mutation assay trial: Evaluation of transferability across 14 laboratories , 2011, Environmental and molecular mutagenesis.

[2]  J. Ashby,et al.  Relative activities of methyl methanesulphonate (MMS) as a genotoxin, clastogen and gene mutagen to the liver and bone marrow of Muta™Mouse mice , 1998, Environmental and molecular mutagenesis.

[3]  M C Cimino,et al.  The in vivo micronucleus assay in mammalian bone marrow and peripheral blood. A report of the U.S. Environmental Protection Agency Gene-Tox Program. , 1990, Mutation research.

[4]  A. Craig,et al.  Oncogenicity by Methyl Methanesulfonate in Male RF Mice , 1968, Science.

[5]  Daniel Krewski,et al.  A Signal-to-Noise Crossover Dose as the Point of Departure for Health Risk Assessment , 2011, Environmental health perspectives.

[6]  L. Müller,et al.  Considerations regarding a permitted daily exposure calculation for ethyl methanesulfonate. , 2009, Toxicology letters.

[7]  G. Speit,et al.  The contribution of excision repair to the DNA effects seen in the alkaline single cell gel test (comet assay). , 1995, Mutagenesis.

[8]  Derek S. Young,et al.  tolerance: An R Package for Estimating Tolerance Intervals , 2010 .

[9]  M. Schuler,et al.  Defining EMS and ENU dose-response relationships using the Pig-a mutation assay in rats. , 2011, Mutation research.

[10]  Kurt Hornik,et al.  The Comprehensive R Archive Network , 2012 .

[11]  Brian A. Nosek,et al.  Power failure: why small sample size undermines the reliability of neuroscience , 2013, Nature Reviews Neuroscience.

[12]  H. Vrieling,et al.  Methyl methanesulfonate-induced hprt mutation spectra in the Chinese hamster cell line CHO9 and its xrcc1-deficient derivative EM-C11. , 1998, Mutation research.

[13]  Michael J. Hendzel,et al.  The γ‐H2A.X: Is it just a surrogate marker of double‐strand breaks or much more? , 2008 .

[14]  Raffaella Corvi,et al.  Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop. , 2008, Mutation research.

[15]  P. White,et al.  Empirical analysis of BMD metrics in genetic toxicology part I: in vitro analyses to provide robust potency rankings and support MOA determinations. , 2016, Mutagenesis.

[17]  R. Schiestl,et al.  Evaluation of the yeast DEL assay with 10 compounds selected by the International Program on Chemical Safety for the evaluation of short-term tests for carcinogens. , 1994, Mutation research.

[18]  Aslihan Gerhold-Ay,et al.  The γH2AX assay for genotoxic and nongenotoxic agents: comparison of H2AX phosphorylation with cell death response. , 2014, Toxicological sciences : an official journal of the Society of Toxicology.

[19]  L. Müller,et al.  MNT and MutaMouse studies to define the in vivo dose response relations of the genotoxicity of EMS and ENU. , 2009, Toxicology letters.

[20]  J. T. Macgregor,et al.  Efficient monitoring of in vivo pig-a gene mutation and chromosomal damage: summary of 7 published studies and results from 11 new reference compounds. , 2012, Toxicological sciences : an official journal of the Society of Toxicology.

[21]  M. Guérard,et al.  Quantitative assessment of the dose–response of alkylating agents in DNA repair proficient and deficient ames tester strains , 2014, Environmental and molecular mutagenesis.

[22]  Gareth J.S. Jenkins,et al.  New approaches to advance the use of genetic toxicology analyses for human health risk assessment , 2015 .

[23]  D. Lovell.  Is tetrachloroethylene genotoxic or not? , 2010, Mutagenesis.

[24]  Masayuki Mishima,et al.  The predominant role of apoptosis in γH2AX formation induced by aneugens is useful for distinguishing aneugens from clastogens. , 2014, Mutation research. Genetic toxicology and environmental mutagenesis.

[25]  S. Dertinger,et al.  The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment , 2010, Environmental and molecular mutagenesis.

[26]  H. Vrieling,et al.  Modulation of the toxic and mutagenic effects induced by methyl methanesulfonate in Chinese hamster ovary cells by overexpression of the rat N-alkylpurine-DNA glycosylase. , 1999, Mutation research.

[27]  T. Singer,et al.  EMS in Viracept--the course of events in 2007 and 2008 from the non-clinical safety point of view. , 2009, Toxicology letters.

[28]  Andrew Teasdale,et al.  ICH M7: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk , 2017 .

[29]  Wout Slob,et al.  Shape and steepness of toxicological dose–response relationships of continuous endpoints , 2014, Critical reviews in toxicology.

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

[31]  G. Jenkins,et al.  Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens. , 2007, Cancer research.

[32]  J. Shaddock,et al.  Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints , 2011, Environmental and molecular mutagenesis.

[33]  Lutz Müller,et al.  Ethyl methanesulfonate toxicity in Viracept--a comprehensive human risk assessment based on threshold data for genotoxicity. , 2009, Toxicology letters.

[34]  K. Witt,et al.  The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup. , 2015, Mutation research. Genetic toxicology and environmental mutagenesis.

[35]  M. Christmann,et al.  MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. , 2007, DNA repair.

[36]  B. Margolin,et al.  Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. , 1987, Mutation research.

[37]  E. Seeberg,et al.  Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts. , 1995, Carcinogenesis.

[38]  T. Singer,et al.  Exposure to Ethylating Agents: Where Do the Thresholds for Mutagenic/Clastogenic Effects Arise? , 2012 .

[39]  F Romagna,et al.  The automated bone marrow micronucleus test. , 1989, Mutation research.

[40]  Krista L Dobo,et al.  Evaluation of the in vivo mutagenicity of isopropyl methanesulfonate in acute and 28‐day studies , 2015, Environmental and molecular mutagenesis.

[41]  Adam D. Thomas,et al.  Theoretical considerations for thresholds in chemical carcinogenesis. , 2015, Mutation research. Reviews in mutation research.

[42]  Adam D. Thomas,et al.  Influence of DNA Repair on Nonlinear Dose-Responses for Mutation , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[43]  L. Hothorn.  Statistical evaluation of toxicological bioassays – a review , 2014 .

[44]  Martin M. Schumacher,et al.  Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations. , 2004, Mutagenesis.

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

[46]  D. Beranek.  Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. , 1990, Mutation research.

[47]  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.

[48]  A. Dobson.  An introduction to generalized linear models , 1990 .

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

[50]  A. Sutter,et al.  Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing. , 2015, Mutation research. Reviews in mutation research.

[51]  A. Natarajan,et al.  The relation between reaction kinetics and mutagenic action of mono-functional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila. , 1979, Mutation research.

[52]  T. Singer,et al.  Possibility of methodical bias in analysis of comet assay studies: Re: DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene. (Mutagenesis, 25, 133-138, 2010). , 2011, Mutagenesis.

[53]  J. T. Macgregor,et al.  IWGT report on quantitative approaches to genotoxicity risk assessment I. Methods and metrics for defining exposure-response relationships and points of departure (PoDs). , 2015, Mutation research. Genetic toxicology and environmental mutagenesis.

[54]  G. Johnson,et al.  Estimating the carcinogenic potency of chemicals from the in vivo micronucleus test. , 2016, Mutagenesis.

[55]  Technical aspects of automatic micronucleus analysis in rodent bone marrow assays , 1994, Cell Biology and Toxicology.

[56]  I. Lemischka,et al.  Clonal and systemic analysis of long-term hematopoiesis in the mouse. , 1990, Genes & development.

[57]  G. Johnson,et al.  No-observed effect levels are associated with up-regulation of MGMT following MMS exposure. , 2008, Mutation research.

[58]  R. W. Lutz,et al.  Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose-response relationships, exemplified for mutagenicity data. , 2009, Mutation research.

[59]  R R Tice,et al.  In vivo rodent erythrocyte micronucleus assay. , 1994, Mutation research.

[60]  S. Mitra.  MGMT: a personal perspective. , 2007, DNA repair.

[61]  J. Heddle,et al.  Mutagenicity of methyl methanesulfonate (MMS) in vivo at the Dlb‐1 native locus and a lacl transgene , 1993, Environmental and molecular mutagenesis.

[62]  T. Helleday,et al.  Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks , 2005, Nucleic acids research.

[63]  J. Bemis,et al.  In vivo mutation assay based on the endogenous Pig‐a locus , 2008, Environmental and molecular mutagenesis.

[64]  E. Zeiger,et al.  Genetic toxicity of procarbazine in bacteria and yeast. , 1979, Mutation research.