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EC number: 911-351-2 | CAS number: -
Table 7.6.1/3:Summary Table of the Experiment I (with and without metabolic activation)
Meanvalues of revertants / Standard deviation (SD)
Salmonella typhimuriumtester strains
4NQO (0.2 µg/plate)
9AA (80 µg/plate)
Notes: ENNG; N-ethyl-N’-nitro-N-nitrosoguanidine; 4NQO; 4-Nitroquinoline-1-oxide; 2AA: 2-aminoanthracene; 9AA: 9-aminoacridine; BP; Benzo(a)pyrene;
In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline and in compliance with GLP, the Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) diluted in Dimethyl sulphoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100 and TA98 and in E. coli WP2 uvr A in the presence and the absence of mammalian metabolic activation (S9) using the direct incorporation or the preincubation method. Six known mutagens (4-nitroquinoline-N-oxide; 9-Aminoacridine; N-ethyl-N-nitro-N-nitrosoguanidine; 2-Aminoanthracene and Benzoapyrene), dissolved in dimethylsulfoxide, were used to check the sensitivity of the test system.
A preliminary study (one plate/concentration) was performed in order to determine the appropriate concentrations for the two independent main studies (3 plates/concentration). In the preliminary study, the bacterial strains TA100 and WP2 uvrA were exposed to the test substance at the following concentrations: 0,0.15,0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test item was freely soluble up to the highest tested concentration. Cytotoxicity, assessed by the decrease in the number of revertants and/or the thinning of the bacterial lawn, was observed in all strains. The test item exhibited weakened background lawns to both strains (TA100 and WP2uvrA) at 5000 µg/plate in the absence and presence of S9-mix. Therefore, 5000 µg/plate was chosen as the highest tested concentration for the main study.
In the first experiment (plate incorporation method; 15, 50, 150, 500, 1500 and 5000 µg/plate) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains at 5000 µg/plate in both the absence and presence of S9-mix. In the second experiment (pre-incubation method with S9 mix, direct plate incorporation without S9 mix; 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) the test item induced a weaker toxic response with weakened bacterial background lawns noted at 5000 µg/plate to TA1537 only in both the absence and presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type and experimental methodology. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.
The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive controls used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
Under the test conditions, the Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl phosphate" (IBAP) did not show any mutagenic activity in the bacterial reverse mutation test using S. typhimurium and E. coli.
This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.
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