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EC number: -
CAS number: -
The results from the in vitro mammalian cell gene mutation test, in
vitro mammalian chromosome aberration test, and bacterial reverse
mutation analysis were negative for genetic toxicity of the test
material. Therefore the test material did not meet the GHS criteria to
be classified as a genetic toxicant.
This study was performed to assess E stage 3 intermediate for its ability to cause gene mutations in vitro cultured mammalian cells L5187Y mouse lymphona cells (TK± -3.7.2C) after being exposed for 3 hours with and without metabolic action and 24 hours without metabolic action respectively using microwell format method. The method was designed to be compatible wth PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 476 'In vitro Cell Gene Mutation Test (2013)'.
L5178Y cells were exposed at 6 doses of 30, 30, 10, 5, 2.5, and 1.25 μg/ml in 3 treatment conditions. The positive and solvent (DMSO) controls were included at the same time in each treatment. The dose volume of each dose group and solvent control were 5μg/ml medium in duplicate. Plating Efficiency (PE) of the cells after exposure was determined and the Relative Total Growth (RTG) was calculated to evaluate toxicity. The Mutant Frequency (MF), Induced Mutant Frequency (IMF) of each culture and the Induced Mutant Frequency of small clone (IMFSC) of the highest dose (10 μg/ml ) and all controls were determined after the inhibition with Trifluorothymidine (TFT). In this test, the results of the solvent and positive control met all validity criteria so the sensitivity of the assay and efficacy of the S9 mixture were validated.
In three treatment conditions, no test item precipitate was observed in any cell culture at all doses before and after incubation.
The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 26%, 91%, 97%, 100% and 86%. The RTG of the cells exposed for 3 hours in the presence of S9 at the doses of 30, 20, 10, 5, 2.5, 1.25μg/ml were 14%, 54%, 85%, 86%, 84% and 103% .The RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 1%, 22%, 60%, 81% and 91%
The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 10 6.
The study was performed to evaluate the ability of E Stage 3 Intermediate to induce reverse mutations in the genome of the histidine requiring test strains of Salmonella typhimurium with and without the metabolic activation system.
Five histidine requiring (his-) mutant tester strains of Salmonella typhimurium including TA97a, TA98, TA100, TA102 and TA1535 were treated with E Stage 3 Intermediate using the standard plate incorporation method and the pre-incubation method at six dose levels, in triplicate, in the presence and absence of co-factor supplemented S9 (S9mix).
Based on the preliminary test results (initial toxicity test) previously performed, six dose levels in the first experiment were selected: 200, 60, 20, 6, 2 and 0.6μg/plate with and without metabolic evaluation. Dimethyl sulfoxide (DMSO) was used as a solvent. The validation experiment was then conducted with the same dose levels and solvent.
In two experiments, the results of the viable count showed that the density of live bacteria in the cultures of each tester strain was within the acceptable range of 0.9~9 x 109 colony forming units (CFU/ml). At the same time, all results of positive and solvent controls met the requiremetns of the test, so the sensitivity of the test and the efficacy of the S9 mix were validated.
Test Item Precipitate:
In the first experiment there was no precipitation at any dose level before and after incubation with and without metabolic activation system. In the validation experiment the same results were obtained as in the first experiment.
In the first experiment cytotoxicity was observed at 200μg/plate dose level of all tester strains in two treated conditions and 60μg/plate dose level of TA102 without metabolic activation. In the validation experiment the same results were obtained as in the first experiment.
In the first experiment with and without metabolic activation, the mean number of revertant colonies at each dose level was less than two times that of the concurrent solvent control in TA1535. In the validation experiment the same results were obtained as in the first experiment.
Under the conditions of this study, the results in both the first experiment and the validation experiment were negative. Thus the test item E Stage 3 Intermediate is considered to be non-mutagenic in the bacterial revrese mutation assay using histidine requiring tester strains of Salmonella typhimurium.
This study was performed to evaluate E stage 3 Intermediate for its ability to cause structural chromosonal aberrations in cultured mammalian cells in three treatment conditions including exposure for three hours with and without metabolic activation and exposure for 24 hours without metablic activation.
In this test, Chinese Hamster Lung (CHL) cell line was treated with E Stage 3 Intermdediate, in three treatment conditions at six dose levels. Positive and solvent controls were included in each condition concurrently.
Based on the results of the preliminary test (initial toxicity test) previously performed, six dose rates of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml medium were included in each treament condition. DMSO was used as solvent. After exposure, all cells were harvested and counted. Then the relative increase in cell count (RICC) was calculated to evaluate the cytotoxicity of the test item. Based on cytotoxicity results, cells at the dose rate with an RICC near 50% (>50%) and two lower doses and cells of all controls in each treatment condition were selected for chromosone preparation. Then microscopic analysis was conducted for chromosonal aberrations.
No precipitation was observed by the naked eye in the cell culture at any dose level in any of the three treatment conditions.
The RICC of the cells exposed for 3 hours in the absence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -29%, 29%, 60%, 82%, 100% and 92%. The RICC of the cells exposed for 3 hours in the presence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -20%, 31%, 73%, 99%, 79% and 89%. The RICC of the cells exposed for 24 hours in the absence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -20%, -4%, 55%, 111%, 103% and 116%.
The results of the microscopic analysis showed that there was no statistically significant difference (P>0.05) in the percentage of cells with structural chromosonal aberration was observed in any treated cells in each treament condition as compared with solvent control. At the same time, statistically significant differences (P< 0.01) were observed in the cells of all positive controls as compared with solvent controls.
The results of this test in all treatment conditions were negative. Therefore, it is considered that the test item E Stage 3 Intermediate did not cause structural chromosonal aberrations in cultured mammalian cells under the conditions of this test.
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