ECVAM retrospective validation of in vitro micronucleus test (MNT)

In the past decade several studies comparing the in vitro chromosome aberration test (CAT) and the in vitro micronucleus test (MNT) were performed. A high correlation was observed in each of the studies (>85%); however, no formal validation for the micronucleus in vitro assay had been carried out. Therefore, a working group was established by the European Centre for the Validation of Alternative Methods (ECVAM) to perform a retrospective validation of the existing data, in order to evaluate the validity of the in vitro MNT on the basis of the modular validation approach. The primary focus of this retrospective validation was on the evaluation of the potential of the in vitro MNT as alternative to the standard in vitro CAT. The working group evaluated, in a first step, the available published data and came to the conclusion that two studies [German ring trial, von der Hude, W., Kalweit, S., Engelhardt, G. et al. (2000) In-vitro micronucleus assay with Chinese hamster V79 cells: results of a collaborative study with 26 chemicals. Mutat. Res., 468, 137–163, and SFTG International Collaborative Study, Lorge, E., Thybaud, V., Aardema, M., Oliver, J., Wataka, A., Lorenzon, G. and Marzin, D. (2006) SFTG International Collaborative Study on in-vitro micronucleus test I. General conditions and overall conclusions of the study. Mutat. Res., 607, 13–36] met the criteria for a retrospective validation according to the criteria previously defined by the working group. These two studies were evaluated in depth (including the reanalysis of raw data) and provided the information required for assessing the reliability (reproducibility) of the test. For the assessment of the concordance between the in vitro MNT and the in vitro CAT, additional published data were considered. Based on this retrospective validation, the ECVAM Validation Management Team concluded that the in vitro MNT is reliable and relevant and can therefore be used as an alternative method to the in vitro CAT. Following peer review, these conclusions were formally endorsed by the ECVAM Scientific Advisory Committee.

[1]  I. Yoshizaki,et al.  Chromosomal aberrations of Don lung cells of Chinese hamster after exposure to vinblastine in vitro. , 1979, Mutation research.

[2]  G. Douglas,et al.  Mutagenic activity of 3 industrial chemicals in a battery of in vitro and in vivo tests. , 1994, Mutation research.

[3]  T. Sofuni,et al.  Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU). , 1999, Mutagenesis.

[4]  K. G. Jensen,et al.  The detection and evaluation of aneugenic chemicals. , 1996, Mutation research.

[5]  M. Sasaki,et al.  Chromosome aberration formation and sister chromatid exchange in relation to DNA repair in human cells. , 1980, Basic life sciences.

[6]  T Sofuni,et al.  A comparative analysis of data on the clastogenicity of 951 chemical substances tested in mammalian cell cultures. , 1988, Mutation research.

[7]  E. Gocke,et al.  Evaluation of the micronucleus test in vitro using chinese hamster cells: Results of four chemicals weakly positive in the in vivo micronucleus test , 1995, Environmental and molecular mutagenesis.

[8]  Daniel Marzin,et al.  SFTG international collaborative study on in vitro micronucleus test I. General conditions and overall conclusions of the study. , 2006, Mutation research.

[9]  S. Roberts,et al.  Extrapolation from in vitro tests to human risk: experience with sodium fluoride clastogenicity. , 1987, Mutation research.

[10]  J. Parry,et al.  The cytochalasin-B micronucleus/kinetochore assay in vitro: studies with 10 suspected aneugens. , 1993, Mutation research.

[11]  Akiko Matsui,et al.  SFTG international collaborative study on in vitro micronucleus test IV. Using CHL cells. , 2006, Mutation research.

[12]  T. Takeuchi,et al.  A comparison of the 8-hydroxydeoxyguanosine, chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes. , 1996, Mutation research.

[13]  Martyn T. Smith,et al.  Characterization of micronuclei induced in human lymphocytes by benzene metabolites. , 1990, Cancer research.

[14]  Daniel Marzin,et al.  SFTG international collaborative study on in vitro micronucleus test V. Using L5178Y cells. , 2006, Mutation research.

[15]  Ü. Egelí,et al.  The Clastogenic effect of Pyrimethamine (Daraprim) on human chromosomes in lymphocyte cultures , 1991, Cell Biology and Toxicology.

[16]  S. Monarca,et al.  In vitro genotoxicity of dimethyl terephthalate. , 1991, Mutation research.

[17]  M. Aardema,et al.  Comparative genotoxicity of quinolone and quinolonyl‐lactam antibacterials in the in vitro micronucleus assay in Chinese hamster ovary cells , 1998, Environmental and molecular mutagenesis.

[18]  W. Hoffman,et al.  A protocol for the in vitro micronucleus test. II. Contributions to the validation of a protocol suitable for regulatory submissions from an examination of 10 chemicals with different mechanisms of action and different levels of activity. , 2002, Mutation research.

[19]  M. Fenech.  Cytokinesis-block micronucleus cytome assay , 2007, Nature Protocols.

[20]  S. McKiernan,et al.  In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances. , 2000, Mutation research.

[21]  E. Zeiger,et al.  Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals , 1989, Environmental and molecular mutagenesis.

[22]  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.

[23]  W. Hoffman,et al.  A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity. , 2002, Mutation research.

[24]  Valérie Zuang,et al.  A Modular Approach to the ECVAM Principles on Test Validity , 2004, Alternatives to laboratory animals : ATLA.

[25]  M. Shelby,et al.  Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro III: Results with 27 chemicals , 1989, Environmental and molecular mutagenesis.

[26]  Ronald D Snyder,et al.  SFTG international collaborative study on in vitro micronucleus test III. Using CHO cells. , 2006, Mutation research.

[27]  G. Obe,et al.  Mutagenicity of methyl 2-benzimidazolecarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies and micronuclei. , 1985, Mutation research.

[28]  J. Parry,et al.  The detection and assessment of the aneugenic potential of environmental chemicals: the European Community Aneuploidy Project. , 1993, Mutation research.

[29]  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.

[30]  Qingguo Huang,et al.  The genotoxicity of substituted nitrobenzenes and the quantitative structure-activity relationship studies. , 1995, Chemosphere.

[31]  A. Osorio,et al.  Genotoxicity of malathion in human lymphocytes assessed using the micronucleus assay in vitro and in vivo: a study of malathion-exposed workers. , 1997, Mutation research.

[32]  R. Pasquini,et al.  Genotoxicity analysis of N,N‐dimethylaniline and N,N‐dimethyl‐p‐toluidine , 1993, Environmental and molecular mutagenesis.

[33]  P. Kasper,et al.  Application of the in vitro rat hepatocyte micronucleus assay in genetic toxicology testing. , 1997, Mutation research.

[34]  M. Ishidate,et al.  The clastogenicity of chemicals in cultured mammalian cells. , 1987, Mutagenesis.

[35]  P. Eckl,et al.  Genotoxic effects of selected peroxisome proliferators. , 1993, Mutation research.

[36]  K. Müller,et al.  An assessment of the in vitro hepatocyte micronucleus assay. , 1993, Mutation research.

[37]  M. Moore,et al.  Mutagenicity of three disinfection by-products: di- and trichloroacetic acid and chloral hydrate in L5178Y/TK +/- (-)3.7.2C mouse lymphoma cells. , 1998, Mutation research.

[38]  C. Tanzarella,et al.  Genotoxic activity of nitrilotriacetic acid in Chinese hamster cells. , 1995, Mutation research.

[39]  S. Venitt,et al.  Chemical transformation of Chinese hamster cells: II. Appearance of marker chromosomes in transformed cells. , 1976, British Journal of Cancer.

[40]  A. Antoccia,et al.  In vitro micronucleus test with kinetochore staining: evaluation of test performance. , 1991, Mutagenesis.

[41]  M. Shelby,et al.  Chromosome aberration and sister chromatid exchange test results with 42 chemicals , 1990, Environmental and molecular mutagenesis.

[42]  V. Mersch-Sundermann,et al.  Genotoxic effects of three Fusarium mycotoxins, fumonisin B1, moniliformin and vomitoxin in bacteria and in primary cultures of rat hepatocytes. , 1997, Mutation research.

[43]  P. Selby,et al.  Lifespan and autopsy findings in the first-generation offspring of X-irradiated male mice. , 1993, Mutation research.

[44]  David Kirkland,et al.  SFTG international collaborative study on in vitro micronucleus test , 2006 .

[45]  T. Sofuni,et al.  Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test. , 1992, Mutation research.

[46]  R. Marcos,et al.  Genotoxicity of the herbicides alachlor and maleic hydrazide in cultured human lymphocytes. , 1996, Mutagenesis.

[47]  A. Bianchi-Santamaria,et al.  Human lymphocyte micronucleus genotoxicity test with mixtures of phytochemicals in environmental concentrations. , 1997, Mutation research.

[48]  C. Körber,et al.  An investigation of micronucleus and mutation induction by oxazepam in mammalian cells. , 1993, Mutagenesis.

[49]  Y. Oshiro,et al.  Chinese hamster ovary cell assays for mutation and chromosome damage: Data from non‐carcinogens , 1991, Journal of applied toxicology : JAT.

[50]  H. Stopper,et al.  Evaluation of the in vitro micronucleus test as an alternative to the in vitro chromosomal aberration assay: position of the GUM Working Group on the in vitro micronucleus test. Gesellschaft für Umwelt-Mutations-forschung. , 1998, Mutation research.

[51]  R. Snyder.  A review and investigation into the mechanistic basis of the genotoxicity of antihistamines. , 1998, Mutation research.

[52]  S Albertini,et al.  Comparative evaluation of the in vitro micronucleus test and the in vitro chromosome aberration test: industrial experience. , 1997, Mutation research.

[53]  D. Busbee,et al.  An accessory protein of DNA polymerase α declines in function with increasing age , 1997 .

[54]  E. Zeiger,et al.  Chromosome aberrations and sister chromatid exchanges in chinese hamster ovary cells: Evaluations of 108 chemicals , 1987, Environmental and molecular mutagenesis.

[55]  A. Martelli,et al.  Testing of p-dichlorobenzene and hexachlorobenzene for their ability to induce DNA damage and micronucleus formation in primary cultures of rat and human hepatocytes. , 1997, Mutagenesis.

[56]  S. Ellard,et al.  Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromes P-450 and microsomal epoxide hydrolase. , 1993, Mutagenesis.

[57]  Véronique Thybaud,et al.  SFTG international collaborative study on in vitro micronucleus test II. Using human lymphocytes. , 2006, Mutation research.

[58]  S Albertini,et al.  Aneuploidy: a report of an ECETOC task force. , 1998, Mutation research.

[59]  S. Madle,et al.  Further evaluation of a modified micronucleus assay with V79 cells for detection of aneugenic effects. , 1993, Mutation research.

[60]  T. Sofuni,et al.  Clastogenicity of 1-nitropyrene, dinitropyrenes, fluorene and mononitrofluorenes in cultured Chinese hamster cells. , 1991, Mutation research.

[61]  S. Morris,et al.  Phenolphthalein induces micronuclei in transgenic human lymphoblastoid cells , 1998, Environmental and molecular mutagenesis.

[62]  E. Zeiger,et al.  Chromosome aberration and sister chromatid exchange tests in chinese hamster ovary cells in vitro. V: Results With 46 Chemicals , 1990, Environmental and molecular mutagenesis.

[63]  E. Rassart,et al.  Induction of micronuclei in vitro by organochlorine compounds in beluga whale skin fibroblasts. , 1999, Mutation research.

[64]  T. Sofuni,et al.  Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens. , 1996, Mutation research.

[65]  M. Fenech,et al.  Report from the In Vitro Micronucleus Assay Working Group , 2003, Mutation research.

[66]  R. Barale,et al.  Genotoxic activity of methyl mercury chloride and dimethyl mercury in human lymphocytes. , 1992, Mutation research.

[67]  M. Kirsch‐Volders.  Towards a validation of the micronucleus test. , 1997, Mutation research.

[68]  E. Zeiger,et al.  Chromosome aberration and sister chromatid exchange tests in chinese hamster ovary cells in vitro: II. Results with 20 chemicals , 1989, Environmental and molecular mutagenesis.

[69]  J. Price,et al.  [News and Views] , 1994, Nature.

[70]  G. Bolcsfoldi,et al.  Comments on the paper 'The non-genotoxicity to rodents of the potent bladder carcinogens o-anisidine and p-cresidine'. , 1992, Mutation research.

[71]  J. Huff,et al.  Benzene-, catechol-, hydroquinone- and phenol-induced cell transformation, gene mutations, chromosome aberrations, aneuploidy, sister chromatid exchanges and unscheduled DNA synthesis in Syrian hamster embryo cells. , 1997, Mutation research.

[72]  T. Ohta,et al.  Rotenone induces aneuploidy, polyploidy and endoreduplication in cultured Chinese hamster cells. , 1991, Mutation research.

[73]  F. Nesslany,et al.  A micromethod for the in vitro micronucleus assay. , 1999, Mutagenesis.