Comet assay evaluation of six chemicals of known genotoxic potential in rats.

As a part of an international validationof the in vivo rat alkaline comet assay( comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene(2-AAF), N-nitrosodimethylamine(DMN), o-anisidine, 1,2-dimethylhydrazinedihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomachof 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogenstested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidineproduced an equivocal result in liver and negative results in stomach, and sodium arsenitewas negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxicresponse. Sodium chloride, a non- genotoxicnon- carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogensproduced clearly positive results in the comet assay, the results obtained for o-anisidineand sodium arsenitein liver and stomachcells are consistent with the known mode of genotoxicityand tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxicmode-of-action and target organ carcinogenicityof 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assayvalidation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals.