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EC number: 276-380-9 | CAS number: 72140-65-9
The mutagenic potential of the test article was evaluated in the Bacterial Reverse Mutation Assay with S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2uvrA in the presence and absence of a metabolic activation system (S9-mix rat liver induced by Phenobarbital and B-napthoflavone). The study was performed in compliance with OECD GLP (1997). The test method was based on OECD No. 471 (1997) and EC No. 440/2008 Part B (2008). The test article was dissolved in dimethyl sulfoxide. A dose range-finding test was performed with concentration up to 5000µg/plate in the absence and presence of metabolic activation in strains TA100 and WP2uvrA. Based on the results of the dose range finding test, the test article was dosed in experiment 1 (direct plate assay) at concentrations of 1.7, 5.4, 17, 52, 164, and 512µg/plate in the absence of S9 and at 5.4, 17, 52, 164, 512, and 1600µg/plate in the presence of S9 in the TA1535, TA1537 and TA98 strains. In order to obtain more information, experiment 2 (pre-incubation assay) exposed TA 1535, TA1537, TA98, and TA100 to concentrations of 10, 25, 50, 100, 200, and 400 μg/plate in the presence and absence of S9. E. coli WP2uvrA was exposed to concentrations of 100, 200, 400, 750, and 1500 μg/plate in the presence and absence of S9. A follow-up third experiment (experiment 3; pre-incubation assay) was performed to evaluate lower concentrations in the absence of S9-mix; TA1535, TA1537, TA98 and TA100 were exposed to concentrations of 0.01, 0.1, 1, 10, and 100 μg/plate in the absence of S9. Strain specific positive controls and negative (vehicle) controls were tested in parallel. All treatments were performed in triplicate. Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the S. typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the E. coli strain contained 15 μg/plate tryptophan. Top agar consisted of Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C. All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 35.8 – 38.8°C). After this period, revertant colonies (His+) for S. typhimurium and (Trp+) for E. coli were counted. In experiment 1 (TA1535, TA1537 and TA98 strains via direct plate assay), precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate. A reduction of the bacterial background lawn and a biologically significant decrease in the number of revertants were observed in all tester strains. No increase in the number of revertants was observed upon treatment with and without metabolic activation. In experiment 2 (TA 1535, TA1537, TA98, and TA100 and WP2uvrA via pre-incubation assay), precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. Moderate to extreme toxicity was observed at all tested concentrations in the absence of S9-mix. Moderate to extreme toxicity was also observed at test item concentrations including or above 50 μg/plate with S9-mix; experiment 3 was performed to evaluate lower concentrations in the absence of S9-mix. In the pre-incubation test, no increase in the number of revertants was observed upon treatment with and without metabolic activation. In experiment 3 (TA1535, TA1537, TA98 and TA100 without S9 via pre-incubation assay), precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. Moderate toxicity was observed at concentrations ≥10 μg/plate. In the pre-incubation test, no increase in the number of revertants was observed upon treatment without metabolic activation. All criteria for a valid test were met as described in the protocol. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the E.coli reverse mutation assay with and without metabolic activation (S9-mix).
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