Genotoxicity testing using the Mutatox™ assay: Evaluation of benzo[a]pyrene as a positive control

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Abstract
In a study on bioassay-directed chemical factionation of sediment extracts, problems were encountered using benzo(a)pyrene (BaP) as a positive control in the Mutatox™ bacterial genotoxicity assay. Genotoxic responses of tests with this compound, prescribed by the Mutatox supplier, could only be measured using supersaturated concentrations that exceeded its aqueous solubility more than 250 times. Additional to three obvious requirements for a positive control of this test system (it should demonstrate that the bacteria are alive and grow after reconstitution, that the bacteria are mutated, and that the bacteria emit light), a positive control should meet two criteria: the metabolic routes of genotoxic response induction for positive control and test compound(s) should be identical, and test concentrations (of both positive control and test compound) should be at environmentally relevant concentrations, i.e., below the aqueous solubilities. As BaP does not meet this last criterion, it does not fully qualify as a positive control in testing genotoxicity of solutes with the Mutatox assay. Testing of other unsubstituted polycyclic aromatic hydrocarbon (PAHS) did not yield another PAH as choice for positive control. Furthermore, the results of our study imply that calculating the bioavailability of BaP from the aqueous solubility alone is not sufficient, as this tends to underestimate the amount of BaP available to either the S9 enzyme system or the bacteria, whereas the interference between toxic and genotoxic effects hampered the interpretation of test results, as variations in toxicity may be interpreted as variations in genotoxicity. Quantitative S9-mediated genotoxicity assessments using the Mutatox test appeared difficult to make, as the two parameters that are proposed by the supplier (CMR, concentration of maximum genotoxic response, and LOEC, lowest observable [genotoxic] effect concentration) showed significant dependency on the sensitivity of the S9 system toward the test compound (CMR) or the initial test compound concentration (LOEC).

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