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Bacterial Mutation Assay

Alkane C6 -C8 (even numbered), 1-sulphonic acid, sodium salt (C6-8 alkane sulfonate) was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain ofEscherichia coli (WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Complementary Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method).

 

Based on the results of the Solubility Test and available information, the test item was dissolved in Distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 or 10 µg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 or 5 μg/plate. In the Initial Mutation Tests and Confirmatory Mutation Tests none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect. In the Confirmatory Mutation Tests inhibitory or cytotoxic effect of the test item was observed inSalmonella typhimuriumTA98 and TA100 bacterial strains at 5000μg/plate concentration without metabolic activation and inSalmonella typhimuriumTA1535 and TA1537 bacterial strains at 5000 and 1581μg/plate concentration without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests. The tests were considered to be valid. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion,C6-8 alkane sulfonatehad no mutagenic activity in the bacterium tester strainsunder the test conditions used in this study.

Chromosome Aberration Test in Mammalian Cells

Test samples of C6-8 alkane sulfonate were assayed in anin vitrocytogenetic study using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate. C6-8 alkane sulfonate was completely dissolved inaqua ad iniectabilia. A correction factor of 1.02 was used as the purity of C6-8 alkane sulfonate was 98% only. The vehicle served as the negative control.

A preliminary cytotoxicity study was conducted to establish the top concentration for the main cytogenetic test.Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µgC6-8 alkane sulfonate/mL medium were employed in an experiment without and with metabolic activation.In this preliminary experiment cytotoxicity was noted at 2500 and 5000 µg C6-8 alkane sulfonate /mL in the experiment with and without metabolic activation (4-h or 24-h exposure). Hence, the highest concentration employed in the main study was 2500 µg C6-8 alkane sulfonate /mL medium in the experiments without and with metabolic activation.

In the main study cytotoxicity was noted in the experiments without and with metabolic activation at the top concentration of 2500 µg C6-8 alkane sulfonate /mL medium. Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with C6-8 alkane sulfonate at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 µg C6-8 alkane sulfonate /mL medium (4-h or 24-h exposure) in the absence of metabolic activation ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).

The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with C6-8 alkane sulfonate at concentrations of 125, 250, 500, 1000, 1500, 2000 or 2500 µg C6-8 alkane sulfonate /mL medium in the presence of metabolic activation in the first and second experiment ranged from 0.5% to 4.0%. These results were within the range of the historical control data (0 - 4%).

The result for the vehicle control cultures was a mean of 1% cells with aberrations (excluding gaps), which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid. No test item-related polyploidy or endoreduplication were noted in the experiments without or with metabolic activation.

In the same test, Mitomycin C and cyclophosphamide induced significant increases in the frequency of damaged cells, which confirmed the validity of this assay.

Conclusion

Under the present test conditions, C6-8 alkane sulfonate, when tested up to a cytotoxic concentration of 2500 µg C6-8 alkane sulfonate /mL medium, in the absence and in the presence of metabolic activation, employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

In Vitro Mammalian Cell Gene Mutation Test

Sodium alkyl sulphate (SAS), a read-across substance for C6-8 alkane sulfonate, was tested in the mouse lymphoma L5178Y mammalian cell gene mutation assay. SAS was tested in both the absence and presence os metabolic activation (S9 -mix) at concentrations of: -S9: 3.125, 6.25, 10, 12.5, 20, 25, 30, 40, 50, 55, 60, 65, 70, 80 and 100 µg/mL, +S9: 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95 µg/mL. The higher dose levels of SAS induced toxic effects in the cells but there was no enhanced mutation rate in the treated or untreated cells, either with or without metabolic activation. The positive controls induced the expected mutagenic response and therefore the test was considered to be valid. Therefore, the test substance was considered to be non-mutagenic.


Short description of key information:
Bacterial mutation assay (Ames test), in vitro chromosome aberration test in mammalian cells, in vitro mammalian cell gene mutation assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, no additional classification is proposed. All three in vitro genotoxicity assays were negative.