The rat S9 microsome fraction is commonly used to assess compound metabolite formation during in vitro genotoxicity assessments. However, methods using S9 have not been standardized for genotoxicity studies, and different experimental methods are used at various facilities. Therefore, this study investigated whether the differences between the two experimental conditions (1) S9 inducers, phenobarbital + beta-naphthoflavones vs. Aroclor 1254 and (2) the plate incubation vs. preincubation method) in the micro-Ames test would affect the results. Nitrosamine and in-house genotoxicity-positive compounds were used with benzopyrene and 2-aminofluorene as positive control compounds. No differences were observed in the genotoxicity results in the groups treated with the positive control. However, the S9 fraction induced by Aroclor 1254 showed higher cytochrome P450 activity than the fraction induced by phenobarbital and beta-naphthoflavone. The incubation method also affected the results; the nitrosamine compounds showed different genotoxicity activity between the plate incorporation method and preincubation method. In-house aminomethyl quinoline compounds also showed different results depending on the S9 type. These results suggest that different inducers and methods induce various metabolic enzyme activities, which may lead to differences in genotoxicity through distinct metabolite production.
Keywords: Aroclor 1254; Genotoxicity; N-nitrosamine; S9 microsome; beta-naphthoflavone; cytochrome P450; phenobarbital.