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2-Nitropropane (2-NP) is a rat liver carcinogen, whilst the 1-isomer is noncarcinogenic in rodents. Although DNA repair tests in the rat liver discriminated clearly between the carcinogenic and the non-carcinogenic isomer, uniformly negative results have been published for the mouse bone marrow micronucleus test (BMMN test) with both isomers. Therefore, the latter assay did not discriminate between the carcinogenic and the noncarcinogenic isomer. To investigate whether this is due to endpoint specificity or organospecificity of 2-NP, studies were carried out in the rat in which micronucleus induction (bone marrow and liver) and unscheduled DNA synthesis (UDS) induction (liver) were measured after oral treatment with either nitropropane isomer. 2-NP induced UDS in the liver whilst the 1-isomer was negative, thus confirming the published studies. In the BMMN test, occasional small increases in the incidence of micronuclei were found for both compounds, but results were interpreted as negative after considering the control background data and the lack of reproducibility. By contrast, the liver micronucleus test revealed a clastogenic effect of 2-NP in the liver. This indicates that 2-NP induces chromosome aberrations as well as DNA repair , but it seems to act organospecifically. For 1-NP a slightly increased incidence of micronuclei was found in the liver, which was accompanied by a markedly increased mitotic index. It therefore remains questionable as to whether this increased micronucleus frequency for 1-NP is an indicator of a clastogenic effect, or whether it is caused by an increased cell proliferation induced by 1-NP. Consequently, it is too early to conclude whether the liver micronucleus assay is able to discriminate between the carcinogenic and non-carcinogenic isomer. However, the results provide further evidence that bone marrow assays are insufficient for the detection of all genotoxic carcinogens . This indicates the need for analysing a second tissue, pzrticularly when negative bone marrow results have been obtained with genotoxins. [ABSTRACT FROM PUBLISHER]

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of in vivo rat alkaline comet assay, we examined p-Chloroaniline, t-butylhydroquinone, and methyl carbamate. All test materials and controls were dosed orally by gavage. p-Chloroaniline produced a statistically significant increase in the mean and median % tail intensity which was also outside of the historical control range in the liver and stomach of Sprague-Dawley rats. t-Butylhydroquinone caused a statistically significant increase in the mean % tail intensity in the liver and stomach and a statistically significant increase in the median % tail intensity in the liver; however, these results are not considered to be biologically significant as all values obtained fell within the current vehicle historical control range and within the negative control range for mean % tail intensity set by the Validation Management Team (VMT) as a requirement for an acceptable assay. Methyl carbamate did not induce a statistically significant change in the mean or median % tail intensity in either liver or stomach., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study., (Copyright © 2015 Elsevier B.V. All rights reserved.)

As a part of the 6th IWGT, an expert working group on the comet assay evaluated critical topics related to the use of the in vivo comet assay in regulatory genotoxicity testing. The areas covered were: identification of the domain of applicability and regulatory acceptance, identification of critical parameters of the protocol and attempts to standardize the assay, experience with combination and integration with other in vivo studies, demonstration of laboratory proficiency, sensitivity and power of the protocol used, use of different tissues, freezing of samples, and choice of appropriate measures of cytotoxicity. The standard protocol detects various types of DNA lesions but it does not detect all types of DNA damage. Modifications of the standard protocol may be used to detect additional types of specific DNA damage (e.g., cross-links, bulky adducts, oxidized bases). In addition, the working group identified critical parameters that should be carefully controlled and described in detail in every published study protocol. In vivo comet assay results are more reliable if they were obtained in laboratories that have demonstrated proficiency. This includes demonstration of adequate response to vehicle controls and an adequate response to a positive control for each tissue being examined. There was a general agreement that freezing of samples is an option but more data are needed in order to establish generally accepted protocols. With regard to tissue toxicity, the working group concluded that cytotoxicity could be a confounder of comet results. It is recommended to look at multiple parameters such as histopathological observations, organ-specific clinical chemistry as well as indicators of tissue inflammation to decide whether compound-specific toxicity might influence the result. The expert working group concluded that the alkaline in vivo comet assay is a mature test for the evaluation of genotoxicity and can be recommended to regulatory agencies for use., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The comet assay can be applied to virtually any tissue and it has been noted that it can be particularly useful in evaluating directly acting genotoxins at their initial site of action. Consequently, it has become relatively common practice to use the stomach comet assay after oral administration to test chemicals that have given positive in vitro genotoxicity results in the absence of metabolic activation. However, to test nontoxic substances up to the limit doses of 1000/2000mg/kg formulations approaching molar concentrations must be used resulting in the stomach mucosa being exposed to excessively high levels. Evidence is beginning to accumulate which shows positive results that do not indicate that potential carcinogenicity may be associated with such high levels of exposure. For pharmaceutical agents, toxicokinetic data are usually available to demonstrate systemic exposure after oral administration. In such cases, it is proposed that exposure of any tissue to levels of the drug substance greater than those that have given positive in vitro results in the absence of metabolic activation is sufficient. However, it is recognised that toxicokinetic data are not available for all chemicals and there are also agents designed not to leave the gastrointestinal tract (GIT). Where it is necessary to examine the GIT, the dose levels selected for examination should cover the likely or intended exposure levels, not necessarily to achieve the maximum tolerated or limit doses, even if the higher doses are required for genotoxicity endpoints in other tissues to be valid. There are usually two or three dose levels in in vivo genotoxicity studies, so when both systemically exposed tissues and the stomach are being examined, it would be possible to use one of the lower doses for the latter without increasing the numbers of animals required. It is important to consider the local concentrations achieved in the stomach or other parts of the GIT in order to avoid the comet assay generating artefactual positive results and it is hoped this will be addressed in the imminent Organisation for Economic Co-operation and Development guideline.

The strategy for testing for genotoxicity covers three main areas, namely gene mutation, chromosome aberration or breakage (clastogenicity), and chromosome loss or gain (aneuploidy). The current generalized strategy consists of assays capable of detecting all of these endpoints using in vitro assays such as the Ames test for detecting gene mutations in bacteria, the human peripheral lymphocyte chromosome aberration (CA) test for detecting clastogenicity, and the in vitro micronucleus test for clastogenicity and aneuploidy. The primary in vivo assay, and generally the only in vivo assay required, is the in vivo rodent bone marrow micronucleus assay. However, there are instances when these assays alone are inadequate and further testing is required, especially in vivo. Historically, the preferred second assay has been the rodent liver unscheduled DNA synthesis assay but recently this has been superseded by the rodent single cell gel electrophoresis or Comet assay. This assay has numerous advantages especially in vivo, where virtually any tissue can be examined. The status of the in vitro comet assay in regulatory testing is much less clear although a preliminary review of data from the assay has shown it to be more specific than other in vitro genotoxicity tests and less prone to false positives.Detailed here are general protocols for both the in vitro and in vivo comet assays which will form the basis of the pending OECD guideline for the assay.