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Genetic toxicity in bacterial strains in vitro (Ames Test)

Caprolactam disulfide was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants (TA 1535, TA 1537, TA 100 and TA 98 ) and Escherichia coli strain WP2uvrA- (Thompson, 2001). The study was performed according to the OECD Guideline 471, EU Method B13/14 and EPA OPPTS 870.5100 without deviations and considered to be of the highest quality (reliability Klimisch 1). The bacteria were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose (15 μg/plate) was used to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. The positive controls N-ethyl-N'-nitro-N-nitrosoguanidine(ENNG, 9-Aminoacridine (9AA) and 4-Nitroquinoline-1-oxide (4NQO) had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. So all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to the majority of the Salmonella tester strains, both with and without S9 -mix, at 5000 μg/plate. No toxicity was observed to Escherichia coli strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. No test material 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 material, either with or without metabolic activation. In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.

Gene mutation in mammalian cells in vitro

The study was performed to investigate the potential of caprolactam disulphide to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Wollny et al., 2011). This experiment was conducted according to OECD Guideline 473 and EU-Method B.17 and according to the principles of good laboratory practice and therefore considered to be of the highest reliability (Klimisch 1). The assay was performed in two independent experiments (two parallel cultures were used throughout the assay), using identical experimental procedures. The cells were exposed to the test item for 4 hours in the first and second experiment with and without metabolic activation. Precipitation of the test item was observed at 22.0 µg/mL in the first main experiment without metabolic activation and at 44.0 µg/mL and above with metabolic activation. In the second experiment precipitation was noted with metabolic activation at 33.0 µg/mL and above. In the second experiment no precipitation was observed without S9 mix.

This shows that the highest concentration applied in the pre-experiment (350 µg/mL) was limited by the solubility properties of the test item in DMSO and aqueous medium. The concentration range of the main experiments was limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. However, appropriate reference mutagens (150 µg/mL EMS and 1.1 µg/mL DMBA) used as positive controls induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this assay.

Chromosome aberrations in mammalian cells in vitro

The substance caprolactam disulphide was investigated for its clastogenic potential in an in vitro mammalian chromosome aberration test with and without metabolic activation using Chinese hamster V79 cells (Nebelung et al., 2011). The study was conducted according to OECD Guideline 473, EU-Method B-10 and EPA OPPTS 870.5375 without deviations and according to the principles of good laboratory practice and considered to be of good quality (reliability Klimisch 1).Chinese hamster V79 cells have been used and as vehicle dimethylsulfoxide was used for test substance solutions and Hanks' balanced salt solution for the positive control substance solutions. Initially the cells were exposed in the absence of S9 mix for 4 hours to concentrations of 3, 6, 12, 18 and 24 µg/mL of the test item. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 12, 18 and 24 µg/mL were harvested 30 hours after the beginning of the treatment. In the presence of S9 mix cells were exposed for 4 hours to concentrations of 7, 14, 28, 38 and 48 µg/mL of test substance. Cultures of all concentrations were harvested 18 hours after the beginning of the treatment. In addition, cells treated with 28, 38 and 48 µg/mL were harvested 30 hours after the beginning of the treatment. Based on cytotoxicity test substance-concentrations were selected for reading of metaphases. Without S9 mix an additional experiment was performed using continuous treatment for 18 hours, harvested at the same time, and the test substance-concentrations of 8, 10 and 12 µg/mL. All test substance-concentrations were read.

Concerning cytotoxicity, effects were observed at 12 µg/mL and above after 4 hours treatment (without S9 -mix)and at 10 µg/mL (and above) after 18 hours treatment. With S9 mix cytotoxic effects were observed at 28 µg/mL and above. Therefore, concentrations of 3, 6 and 18 µg/mL test substance (4 hours treatment) were chosen for reading in the absence of S9 mix. In the presence of S9 mix 7, 14 and 28 µg/mL of test substance were employed. All of these cultures harvested 18 hours after the beginning of the treatment were included. In addition, cultures treated in the absence of S9 mix with 18 µg/mL and harvested 30 hours after the beginning of the treatment were used. The same was true for cultures treated in the presence of S9 mix with 28 µg/mL. None of the cultures treated with the test substance in the absence and in the presence of S9 mix showed biologically relevant increased numbers of aberrant metaphases. However, the positive controls mitomycin C and cyclophosphamide induced clastogenic effects and demonstrated the sensitivity of the test system and the activity of the used S9 mix.Based on this test, the test item is considered not to be clastogenic for mammalian cells in vitro.


Justification for selection of genetic toxicity endpoint
No study was selected, since all three in vitro studies were negative

Short description of key information:
1) Thompson, 2001, Salmonella/microsome test, doses up to 5000 µg/plate, non-mutagenic
2) Wollny, 2011, HPRT-test using Chinese hamster V79 cells, doses up to 350 µg/mL, non-mutagenic
3) Nebelung, 2011, mammalian chromosome-aberration test using Chinese hamster V79 cells, doses up to 48 µg/mL, non-clastogenic

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test material does not meet the criteria for classification and will not require labelling as a mutagen in accordance with European regulation (EC) No 1272/2008.