GENOTOXICITY TESTING USING THE MUTATOX(TM) ASSAY - EVALUATION OF BENZO[A]PYRENE AS A POSITIVE CONTROL

In a study on bioassay-directed chemical fractionation of sediment ext racts, problems were encountered using benzo(a)pyrene (BaP) as a posit ive control in the Mutatox((TM)) bacterial genotoxicity assay. Genotox ic responses of tests with this compound, prescribed by the Mutatox su pplier, could only be measured using supersaturated concentrations tha t exceeded its aqueous solubility more than 250 times. Additional to t hree obvious requirements for a positive control of this test system ( it should demonstrate that the bacteria are alive and grow after recon stitution, that the bacteria are mutated, and that the bacteria emit l ight), a positive control should meet two criteria: the metabolic rout es of genotoxic response induction for positive control and test compo und(s) should be identical, and test concentrations (of both positive control and test compound) should be at environmentally relevant conce ntrations, i.e., below the aqueous solubilities. As BaP does not meet this last criterion, it does not fully qualify as a positive control i n testing genotoxicity of solutes with the Mutatox assay. Testing of o ther unsubstituted polycyclic aromatic hydrocarbon (PAHS) did not yiel d another PAH as choice for positive control. Furthermore, the results of our study imply that calculating the bioavailability of BaP from t he aqueous solubility alone is not sufficient, as this tends to undere stimate the amount of BaP available to either the S9 enzyme system or the bacteria, whereas the interference between toxic and genotoxic eff ects hampered the interpretation of test results, as variations in tox icity may be interpreted as variations in genotoxicity. Quantitative S E-mediated genotoxicity assessments using the Mutatox test appeared di fficult to make, as the two parameters that are proposed by the suppli er (CMR, concentration of maximum genotoxic response, and LOEC, lowest observable [genotoxic] effect concentration) showed significant depen dency on the sensitivity of the S9 system toward the test compound (CM R) or the initial test compound concentration (LOEC).