Improvement of in vivo genotoxicity assessment: combination of acute tests and integration into standard toxicity testing.

A working group convened at the 2009 5th IWGT to discuss possibilities for improving in vivo genotoxicity assessment by investigating possible links to standard toxicity testing. The working group considered: (1) combination of acute micronucleus (MN) and Comet assays into a single study, (2) integration of MN assays into repeated-dose toxicity (RDT) studies, (3) integration of Comet assays into RDT studies, and (4) requirements for the top dose when integrating genotoxicity measurements into RDT studies. The working group reviewed current requirements for in vivo genotoxicity testing of different chemical product classes and identified opportunities for combination and integration of genotoxicity endpoints for each class. The combination of the acute in vivo MN and Comet assays was considered by the working group to represent a technically feasible and scientifically acceptable alternative to conducting independent assays. Two combination protocols, consisting of either a 3- or a 4-treament protocol, were considered equally acceptable. As the integration of MN assays into RDT studies had already been discussed in detail in previous IWGT meetings, the working group focussed on factors that could affect the results of the integrated MN assay, such as the possible effects of repeated bleeding and the need for early harvests. The working group reached the consensus that repeated bleeding at reasonable volumes is not a critical confounding factor for the MN assay in rats older than 9 weeks of age and that rats bled for toxicokinetic investigations or for other routine toxicological purposes can be used for MN analysis. The working group considered the available data as insufficient to conclude that there is a need for an early sampling point for MN analysis in RDT studies, in addition to the routine determination at terminal sacrifice. Specific scenarios were identified where an additional early sampling can have advantages, e.g., for compounds that exert toxic effects on hematopoiesis, including some aneugens. For the integration of Comet assays into RDT studies, the working group reached the consensus that, based upon the limited amount of data available, integration is scientifically acceptable and that the liver Comet assay can complement the MN assay in blood or bone marrow in detecting in vivo genotoxins. Practical issues need to be considered when conducting an integrated Comet assay study. Freezing of tissue samples for later Comet assay analysis could alleviate logistical problems. However, the working group concluded that freezing of tissue samples can presently not be recommended for routine use, although it was noted that results from some laboratories look promising. Another discussion topic centred around the question as to whether tissue toxicity, which is more likely observed in RDT than in acute toxicity studies, would affect the results of the Comet assay. Based on the available data from in vivo studies, the working group concluded that there are no clear examples where cytotoxicity, by itself, generates increases or decreases in DNA migration. The working group identified the need for a refined guidance on the use and interpretation of cytotoxicity methods used in the Comet assay, as the different methods used generally lead to inconsistent conclusions. Since top doses in RDT studies often are limited by toxicity that occurs only after several doses, the working group discussed whether the sensitivity of integrated genotoxicity studies is reduced under these circumstances. For compounds for which in vitro genotoxicity studies yielded negative results, the working group reached the consensus that integration of in vivo genotoxicity endpoints (typically the MN assay) into RDT studies is generally acceptable. If in vitro genotoxicity results are unavailable or positive, consensus was reached that the maximum tolerated dose (MTD) is acceptable as the top dose in RDT studies in many cases, such as when the RDT study MTD or exposure is close (50% or greater) to an acute study MTD or exposure. Finally, the group agreed that exceptions to this general rule might be acceptable, for example when human exposure is lower than the preclinical exposure by a large margin.

[1]  Zoryana Cammerer,et al.  In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals. , 2007, Mutation research.

[2]  J. Högel,et al.  Characterization of the genotoxic potential of formaldehyde in V79 cells. , 2007, Mutagenesis.

[3]  Catherine C. Smith,et al.  Recommendations for design of the rat comet assay. , 2008, Mutagenesis.

[4]  P. Clay.  Assessment of the genotoxicity of trichloroethylene and its metabolite, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), in the comet assay in rat kidney. , 2007, Mutagenesis.

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

[6]  Zoryana Cammerer,et al.  Comparison of the peripheral blood micronucleus test using flow cytometry in rat and mouse exposed to aneugens after single-dose applications. , 2007, Mutagenesis.

[7]  Marie Z. Vasquez.  Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation , 2009, Mutagenesis.

[8]  Trevelyan Square,et al.  Recommendations for conducting the in vivo alkaline Comet assay , 2003 .

[9]  J. T. Macgregor,et al.  Integration of mutation and chromosomal damage endpoints into 28-day repeat dose toxicology studies. , 2010, Toxicological sciences : an official journal of the Society of Toxicology.

[10]  R. Schlegel,et al.  The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening. , 1984, Mutation research.

[11]  J. T. Macgregor,et al.  In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring , 2000, Environmental and Molecular Mutagenesis.

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

[13]  Y. Miyamoto,et al.  An in vivo comet assay of multiple organs (liver, kidney and bone marrow) in mice treated with methyl methanesulfonate and acetaminophen accompanied by hematology and/or blood chemistry. , 2008, The Journal of toxicological sciences.

[14]  Andreas Czich,et al.  Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies. , 2010, Mutation research.

[15]  W. Slikker,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes IV: an index of chromosomal damage in the rhesus monkey (Macaca mulatta). , 2008, Toxicological sciences : an official journal of the Society of Toxicology.

[16]  F. Nesslany,et al.  In vivo Comet assay on isolated kidney cells to distinguish genotoxic carcinogens from epigenetic carcinogens or cytotoxic compounds. , 2007, Mutation research.

[17]  S. Monarca,et al.  In vivo studies on genotoxicity of pure and commercial linuron. , 1997, Mutation research.

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

[19]  N. Yajima,et al.  Comparative studies in induction of micronuclei by three genetically recombinant and urinary human erythropoietins. , 1993, Mutagenesis.

[20]  S. Windebank,et al.  Induction of micronuclei in rat bone marrow and peripheral blood following acute and subchronic administration of azathioprine. , 1993, Mutation research.

[21]  L. Poulain,et al.  Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis. , 1999, Cytometry.

[22]  Raffaella Corvi,et al.  Reduction of use of animals in regulatory genotoxicity testing: Identification and implementation opportunities-Report from an ECVAM workshop. , 2009, Mutation research.

[23]  T Sofuni,et al.  Report of the IWGT working group on strategy/interpretation for regulatory in vivo tests II. Identification of in vivo-only positive compounds in the bone marrow micronucleus test. , 2007, Mutation research.

[24]  F. Beland,et al.  DNA adduct measurements and tumor incidence during chronic carcinogen exposure in rodents. , 1994, Environmental health perspectives.

[25]  N. Yajima,et al.  Induction of micronucleated erythrocytes by recombinant human erythropoietin. , 1993, Mutagenesis.

[26]  J. T. Macgregor,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: I. Intra- and interlaboratory comparison with microscopic scoring. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[27]  A. Wakata,et al.  Evaluation of the rodent micronucleus assay by a 28‐day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)–Mammalian Mutagenicity Study Group (MMS) , 2001, Environmental and molecular mutagenesis.

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

[29]  J. T. Macgregor,et al.  Sensitivity of the erythrocyte micronucleus assay: dependence on number of cells scored and inter-animal variability. , 2007, Mutation research.

[30]  Thomas Brüning,et al.  Renal toxicity after chronic inhalation exposure of rats to trichloroethylene. , 2002, Toxicology letters.

[31]  J. Li,et al.  The micronucleus test and erythropoiesis. Effects of erythropoietin and a mutagen on the ratio of polychromatic to normochromatic erythrocytes (P/N ratio). , 1989, Mutagenesis.

[32]  K. Witt,et al.  Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol. , 2010, The Journal of toxicological sciences.

[33]  M. Hayashi,et al.  In vivo rodent micronucleus assay: protocol, conduct and data interpretation. , 2000, Mutation research.

[34]  M. Garriott,et al.  The in vivo rat micronucleus test: integration with a 14-day study. , 1995, Mutation research.

[35]  G. Krishna,et al.  Principles and practices of integrating genotoxicity evaluation into routine toxicology studies: A pharmaceutical industry perspective , 1998, Environmental and molecular mutagenesis.

[36]  J. T. Macgregor,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes III. An efficient method of monitoring chromosomal damage in the beagle dog. , 2007, Toxicological sciences : an official journal of the Society of Toxicology.

[37]  M. Shelby,et al.  The in vivo erythrocyte micronucleus test: measurement at steady state increases assay efficiency and permits integration with toxicity studies. , 1990, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[38]  K. Mitsumori,et al.  Absence of in vivo genotoxicity and liver initiation activity of dicyclanil. , 2003, The Journal of toxicological sciences.

[39]  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.

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

[41]  Stephen D Dertinger,et al.  In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test. , 2007, Mutation research.

[42]  K. Witt,et al.  No increases in biomarkers of genetic damage or pathological changes in heart and brain tissues in male rats administered methylphenidate hydrochloride (Ritalin) for 28 days , 2009, Environmental and molecular mutagenesis.

[43]  J. T. Macgregor,et al.  Integration of cytogenetic assays with toxicology studies , 1995, Environmental and molecular mutagenesis.

[44]  Junichiro Saito,et al.  Flow cytometric analysis of micronuclei in peripheral blood reticulocytes: II. An efficient method of monitoring chromosomal damage in the rat. , 2006, Toxicological sciences : an official journal of the Society of Toxicology.

[45]  A. Buschini,et al.  Modulating effects of humic acids on genotoxicity induced by water disinfectants in Cyprinus carpio. , 2005, Mutation research.

[46]  M D Shelby,et al.  Micronucleated erythrocyte frequency in peripheral blood of B6C3F1 mice from short‐term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program , 2000, Environmental and molecular mutagenesis.

[47]  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.

[48]  Makoto Hayashi,et al.  Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS·MMS , 1998, Environmental and molecular mutagenesis.

[49]  A. Zijno,et al.  Analysis of micronuclei and DNA single-strand breaks in mouse splenocytes and peripheral lymphocytes after oral administration of tetramethylthiuram disulfide (thiram). , 1998, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[50]  R R Tice,et al.  Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop. , 2003, Mutagenesis.

[51]  The suitability of rat peripheral blood in subchronic studies for the micronucleus assay. , 1995, Mutation research.

[52]  M. Cimino,et al.  An evaluation of the mode of action framework for mutagenic carcinogens case study: Cyclophosphamide , 2008, Environmental and molecular mutagenesis.

[53]  Y. Sasaki,et al.  Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline single cell gel electrophoresis (comet) assay in multiple mouse organs. , 1998, Mutation research.

[54]  Stephen D Dertinger,et al.  Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. , 2003, Toxicological sciences : an official journal of the Society of Toxicology.

[55]  D. Salvadori,et al.  No Relationship between Subchronic Fluoride Intake and DNA Damage in Wistar Rats , 2004, Caries Research.