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The optimal use of historical control data for the interpretation of genotoxicity results was discussed at the 2009 International Workshop on Genotoxicity Testing (IWGT) in Basel, Switzerland. The historical control working group focused mainly on negative control data although positive control data were also considered to be important. Historical control data are typically used for comparison with the concurrent control data as part of the assay acceptance criteria. Historical control data are also important for providing evidence of the technical competence and familiarization of the assay at any given laboratory. Moreover, historical control data are increasingly being used to aid in the interpretation of genetic toxicity assay results. The objective of the working group was to provide generic advice for historical control data that could be applied to all assays rather than to give assay-specific recommendations. In brief, the recommendations include

At the 2009 International Workshop on Genotoxicity Testing in Basel, an expert group gathered to provide guidance on suitable follow-up tests to describe risk when basic in vivo genotoxicity tests have yielded positive results. The working group agreed that non-linear dose-response curves occur in vivo with at least some DNA-reactive agents. Quantitative risk assessment in such cases requires the use of (1) adequate data, i.e., the use of all available data for the selection of reliable in vivo models to be used for quantitative risk assessment, (2) appropriate mathematical models and statistical analysis for characterizing the dose-response relationships and allowing the use of quantitative and dose-response information in the interpretation of results, (3) mode of action (MOA) information for the evaluation and analysis of risk, and (4) reliable assessments of the internal dose across species for deriving acceptable margins of exposure and risk levels. Hence, the elucidation of MOA and understanding of the mechanism underlying the dose-response curve are important components of risk assessment. The group agreed on the need for (i) the development of in vivo assays, especially multi-endpoint, multi-species assays, with emphasis on those applicable to humans, and (ii) consensus about the most appropriate mathematical models and statistical analyses for defining non-linear dose-responses and exposure levels associated with acceptable risk.

Cytosine arabinoside (a nucleoside analogue that inhibits the gap-filling step of excision repair), vinblastine (an aneugen that inhibits tubulin polymerisation), 5-fluorouracil (a nucleoside analogue with a steep response profile), and 2-aminoanthracene (a metabolism-dependent reference genotoxin) were tested in the in vitro micronucleus assay with L5178Y mouse lymphoma cells, without cytokinesis block. The four chemicals were independently evaluated in two Sanofi Aventis laboratories, one of which used an image analyser to score micronuclei, while the other scored micronucleated cells manually. Very similar results were obtained in the two laboratories, highlighting the robustness of the assay. The four test chemicals induced significant increases in the incidence of micronucleated cells at concentrations that produced no more than a 55 ± 5% reduction in survival growth, as measured with the three parameters recommended in the draft OECD Test Guideline on In Vitro Mammalian Cell Micronucleus Test (MNvit) for chemical testing, namely the relative increase in cell counts, relative population doubling, and the relative cell count. These results support the premise that the relative increase in cell counts and relative population doubling, that take into account both cell death and cytostasis, are appropriate measures of survival growth reduction in the in vitro micronucleus test conducted in the absence of cytokinesis block, as recommended in MNvit.

Gene expression profiling technology is expected to advance our understanding of genotoxic mechanisms involving direct or indirect interaction with DNA. We exposed human lymphoblastoid TK6 cells to 14 anticancer drugs (vincristine, paclitaxel, etoposide, daunorubicin, camptothecin, amsacrine, cytosine arabinoside, hydroxyurea, methotrexate, 5-fluorouracil, cisplatin, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), and bleomycin) for 4-h and examined them immediately or after a 20-h recovery period. Cytotoxicity and genotoxicity, respectively, were evaluated by cell counting and by in vitro micronucleus assay at 24h. Effects on the cell cycle were determined by flow cytometry at 4 and 24h. Gene expression was profiled at both sampling times by using human Affymetrix U133A GeneChips (22K). Bioanalysis was done with Resolver/Rosetta software and an in-house annotation program. Cell cycle analysis and gene expression profiling allowed us to classify the drugs according to their mechanisms of action. The molecular signature is composed of 28 marker genes mainly involved in signal transduction and cell cycle pathways. Our results suggest that these marker genes could be used as a predictive model to classify genotoxins according to their direct or indirect interaction with DNA.

This study on the in vitro micronucleus assay, comprising 11 laboratories using human lymphocytes, was coordinated by an organizing committee supported by the SFTG (the French branch of the European Environmental Mutagen Society). Nine coded substances were assessed for their ability to induce micronuclei in human lymphocytes in vitro, mitomycin C being used as a positive control. Cultures were exposed to the test substances for a short (early or late) time or for a long time, followed by a short or long recovery period, in the presence of cytochalasin B. Each chemical was evaluated, generally in two laboratories, using three treatment schedules at least twice. The data were assessed for acceptability, and then classified as negative, positive or equivocal. Two of seven genotoxic compounds, namely colchicine and bleomycin, clearly induced micronuclei. Reproducible results were difficult to obtain for some substances, which tended to be those acting at specific stages of the cell cycle. Cytosine arabinoside, diethylstilboestrol and 5-fluorouracil were classified as equivocal. Urethane and thiabendazole were classified as negative. The two presumed non-genotoxic compounds, mannitol and clofibrate, did not induce micronuclei. Repeat testing, exposing cells at both an early and late time after mitogenic stimulation, was needed to detect substances classified as equivocal. These results show the importance of achieving sufficient inhibition of nuclear division to avoid the possibility of missing an effect. The evaluation of micronuclei in mononucleated as well as binucleated cells was particularly useful to detect aneugens. There were no false positive results using lymphocytes, indicating a high specificity. It is concluded that the clastogenic or aneugenic potential in vitro of the substances tested was correctly identified in this study, but that refining the protocol to take into account factors such as the stages of the cell cycle exposed to the compound, or the duration of recovery would be likely to improve the sensitivity of detection using lymphocytes.

7H-Dibenzo[c,g]carbazole (DBC) is an environmental pollutant that produces DNA adducts and tumors in mouse liver and skin following subcutaneous injection and topical application. The two synthetic derivatives 5,9-dimethyl-DBC (DMDBC) and N7-methyl-DBC (NMDBC) induce tissue-specific lesions. DNA adducts and tumors are observed only in liver following exposure to DMDBC and only in skin following exposure to NMDBC. We used the positive selection MutaMouse model to measure the induction of mutations in the two target organs, 28 days after a single subcutaneous injection or topical application of DBC, DMDBC and NMDBC. In liver, DBC and DMDBC induced 30- to 50-fold increases in mutant frequency (MF), while NMDBC had only a weak effect, regardless of the route of administration. After topical application, DBC and NMDBC produced 3.4- to 7.9-fold increases in MF in skin, while DMDBC had a weak effect. After subcutaneous injection, the three compounds had no or weak effect in skin. This study shows gene mutations arise in the respective target organs in which primary DNA damage and tumors are observed. These results illustrate the relevance of the MutaMouse model for testing organ-specific mutagens.

As part of the Germ Cell Collaborative Study, we used the positive-selection Muta™Mouse model to evaluate the effects of two direct alkylating agents, ethylnitrosourea (ENU) and methyl methanesulfonate (MMS), on male germ cells. The LacZ mutation frequency in seminiferous tubule cells and epididymis spermatozoa was measured 3, 14, 25 and 50 days after a single intraperitoneal (i.p.) administration of 150 mg/kg ENU and 3 and 14 days after a single i.p. administration of 40 mg/kg MMS. Three and 14 days after ENU treatment, the mutation frequency was slightly but significantly increased in seminiferous tubule cells (3.5- and 3.6-fold, respectively), while it remained unchanged in epididymis spermatozoa. After 25 and 50 days, time-dependent increase in the mutation frequency was observed in seminiferous tubule cells (8.9- and 14.3-fold, respectively) and epididymis spermatozoa (3.4- and 7.9-fold, respectively), confirming the sensitivity of premeiotic cells to the mutagenic activity of ENU. Three and 14 days after MMS administration, the mutation frequency remained unchanged in seminiferous tubule cells and epididymis spermatozoa. The inability of Muta™Mouse model to reveal the mutagenic activity of MMS was confirmed in bone marrow cells, 14 days after treatment. These data indicate that the Muta™Mouse model can be used to detect the induction of gene mutations but not chromosome damage in germ cells.

The Ames test is now widely used as a short-term test for the detection of mutagens. Different strains are available with various genetic characteristics, and in the past decade various authors have recommended different associations of strains to give maximum detection potential. However, few studies have been done to compare the sensitivity of individual strains towards a wide range of compounds in a single study. In order to define the best association of strains for screening or regulatory purpose, we have tested 103 direct mutagens (reference genotoxins or in-house compounds) on 7 strains of Salmonella typhimurium: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102. 126 different associations of strains have been studied in terms of sensitivity and percentage overlap. Optimum associations of 2, 3, 4 or 5 strains included strains both with and without plasmid pKM101. However, the specificity of detection is greatly diminished by the presence of plasmid pKM101 in the strain, as shown by the high degree of overlap in associations constituted entirely of strains containing the plasmid. The association of strains TA1538 and TA100 detected 86% of the chemicals tested and is therefore recommended for large-scale screening. A rate of detection of 100% was obtained when 6 strains were used. The best associations of 4 and 5 strains, which detected 97 and 99% chemicals respectively, all contained strains TA1537, TA1538 and TA102. Finally, the associations of 4 strains (TA1537, TA1538, TA100, TA102) or 5 strains (TA1535, TA1537, TA1538, TA97, TA102) seemed well adapted to the optimum detection of mutagenic compounds.