The influence of exogenous metabolism on the specificity of in vitro mammalian genotoxicity tests

A two-part study was designed to determine whether the inclusion of the rodent liver 'S9' exogenous metabolic activating system contributes to the generation of misleading positive results by the regulator-required in vitro mammalian genotoxicity tests. The mono-oxygenase enzymes in S9 produce direct-acting DNA-reactive electrophiles, and are included in in vitro genotoxicity tests to enhance the detection of substances which only become genotoxic following metabolism. However, as the S9 system lacks 'detoxifying' phase 2 factors it was hypothesised that increased chemical metabolism per se may lead to an increase in irrelevant S9 test outcomes in safety assessment. To test this, 89 compounds with positive or negative carcinogenicity data were identified, which produced negative Ames test data (+/- S9), and only produced positive in vitro mammalian test data in the presence of S9. This allowed a determination of whether or not misleading predictions of carcinogenicity by the in vitro mammalian tests were more or less prevalent in the presence of S9. A subset of these compounds was then tested with and without S9 in the GADD45a-GFP genotoxicity test, in order to determine whether misleading in vitro mammalian positive results were generally more prevalent with S9, or reflected particular tests' liabilities. This study suggests that the use of S9 metabolic activation in in vitro genotoxicity tests does not increase the prevalence of misleading positive results in in vitro mammalian genotoxicity assays, at least amongst Ames negative compounds.