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Key value for chemical safety assessment

Additional information

Gene mutation

In a key study, the potential of the substance to induce reverse mutation in bacteria was assessed using four strains of Salmonella typhimurium and Escherichia Coli WP2 uvrA according to the OECD guideline 471.The study was conducted in compliance with Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without metabolic activation. Both experiments were performed according to the direct plate incorporation. After 48 hours of incubation at 37°C, the revertant colonies were scored.

The preliminary assays showed that the test substance was freely soluble and non-toxic in all tested strains. The highest dose-level was 5000 µg /plate with a dose range of 50 to 5000 µg/plate. In the two independent assays, a white precipitate was observed in the petri plates at 5000 µg/plate. No toxicity was noted at any dose-level in any strain. The test item did not induce any noteworthy increase in the number of revertants, in any of the strains, either with or without metabolic activation.

Under these experimental conditions, the substance did not show any mutagenic activities in the bacterial reverse mutation test.

The substance was tested for mutations at the Thymidine-kinase (TK) locus in L5178Y mouse lymphoma cells according to OECD guideline 476. The study was conducted in compliance with Good Laboratory Practices.

In this key study, the test material was tested in two independent experiments, both with and without metabolic activation. Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main experiments was based on the level of toxicity (decrease in Relative Total Growth -RTG), according to the criteria specified in the international guidelines.

In the first experiment, cells were exposed to the test or control materials (with or without metabolic activation) for 4 hours. Six concentrations, ranging from 300 to 5000 µg/mL, were tested.

In the second experiment, cells were exposed to the test or control items for 24 hours without metabolic activation and for 4 hours with metabolic activation. Six concentrations, ranging from 150 to 1500 µg/ml and from 300 to 3600 µg/ml were tested with and without metabolic activation respectively.

At the end of the 4 -and 24 -hour treatment periods, a precipitate was noted in the culture medium at dose levels equal or higher than 312.5 µg/ml and 156.25 µg/ml, respectively. Cytotoxicity was observed in both experiments with and without S9 mix.

No noteworthy increase in the mutation frequency was induced after 4h treatment with or without metabolic activation. After 24 h treatment, there was small but statistically significant increase in mutant frequency. The response observed was very modest, at only one cytotoxic dose level and did not exceed the Global Evaluation Factor. The mutant frequency value was well within the acceptable range for L5178Y cells and involved a vehicle control that was at the lower end of the acceptable range. Therefore, the observed response was considered to have no toxicological significance.

It was concluded that the substance did not induce mutations at the thymidine-kinase locus in L5178Y mouse lymphoma cells.

Chromosomal aberration assay

In this key study, the potential of the substance to induce chromosomal aberrations in vitro was investigated in cultured human lymphocytes according to OECD guideline 476.The study was conducted in compliance with Good Laboratory Practices.

The test material was tested in two independent experiments, both with and without metabolic activation. The highest dose-level for treatment was selected on the basis of pH, osmolality and solubility. Any toxicity indicated by the reduction of Mitotic Index was also taken into account. In the first experiment, lymphocyte cultures were exposed to the test or control materials with or without metabolic activation for 4 hours. Cells were harvested 20 hours after the end of the exposure period. The second experiment was performed as follows: without metabolic activation, cells were exposed continously to the test or control materials until harvest; with metabolic activation, cells received the same treatment as in the first experiment.

The maximum dose-level selecting for metaphase analysis (625 µg/ml) was based on the lowest precipitating dose-level which was also the maximum dose-level that could be evaluated. Without metabolic activation, the test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in the two separate experiments. With metabolic activation, the test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in the first experiment. In the second experiment, a statistically significant increase in the frequency of cells with aberrations was observed at 625 µg/ml. However, although the increase excedeed the historical maxima, it was small, not reproduced in the first experiment and therefore considered to have no toxicological significance. In either of the experiments, the test material did not induce significant increase in the number of polyploid cells.

The substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.


Justification for selection of genetic toxicity endpoint
None selected, all tests were negative

Short description of key information:
The substance produced no genetic changes in standard in vitro tests using bacterial and mammalian cells and performed according to OECD guidelines and GLP. No gene mutations were induced in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. Coli WP2 uvrA. No chromosomal aberrations were induced in cultured human lymphocytes and no increases in gene mutations were observed in mouse lymphoma L5178Y cells.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All in vitro genetic toxicity tests were negative. On the basis of these results and according to regulation (EC) N° 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, no classification is warranted with respect to genetic toxicity.