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Additional information

Three in vitro genetic toxicity studies have been conducted on the test material, as follows:

in vitro gene mutation study in bacteria: The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the OECD Guideline 471. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation and pre-incubation methods at up to 7 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the experiment was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The range was amended slightly and ranged between 5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. In the first experiment (plate incorporation methodology), the test item caused no visible reduction in the growth of the bacterial background lawns at any dose level either in the absence or presence of S9-mix. In the second experiment (pre-incubation methodology), the test item induced toxicity as weakened bacterial background lawns of all of the Salmonella strains dosed in the absence of S9-mix from 1500 µg/plate and at 5000 µg/plate to the Salmonella strains dosed in the presence of S9-mix and Escherichia coli strain WP2uvrA dosed both in the absence and presence of S9-mix. 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 test item was considered to be non-mutagenic under the conditions of the test.

in vitro micronucleus study: This report describes the results of an in vitro study for the detection of structural chromosome aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations. The method was designed to be compatible with OECD Guideline 473. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at 3 dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. in Experiment 1, 4 h in the presence of an induced rat liver homogenate metabolising system (S9), at a 2 % final concentration with cell harvest after a 20-h expression period and a 4 h exposure in the absence of metabolic activation (S9) with a 20-h expression period. In Experiment 2, the 4 h exposure with addition of S9 was repeated (using a 1 % final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 h.

The dose levels used in the main experiments were selected using the data from the preliminary toxicity test and were as follows:

Group

Final concentration of test item (µg/mL)

4(20)-h without S9

90, 180, 360, 540, 720, 900

4(20)-h with S9 (2 %)

180, 360, 540, 720, 810, 900

24-h without S9

180, 360, 540, 720, 810, 900

4(20)-h with S9 (1 %)

180, 360, 540, 720, 900, 1080

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of 2 separate experiments, using a dose range that was limited by the onset of a greasy oily precipitate and included a dose level that exceeded 50 % mitotic inhibition. The test item was considered to be non-clastogenic to human lymphocytes in vitro.

in vitro gene mutation study in mammalian cells: The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline 476. Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (2% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test. For Experiments 1 the dose range was 25 to 350 μg/mL in the absence of metabolic activation and 75 to 1200 μg/mL in the presence of metabolic activation. In Experiment 2 the dose range was 25 to 400 μg/mL in the absence of metabolic activation and 37.5 to 1050 μg/mL in the presence of metabolic activation. The maximum dose levels used in the Mutagenicity Test was limited by test item induced toxicity. A precipitate of the test item was observed at and above 750 μg/mL in the presence of metabolic activation only.The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.


Short description of key information:
in vitro gene mutation study in bacteria: The test item was determined to be non-mutagenic under the conditions of this test.

in vitro micronucleus study: The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

in vitro gene mutation study in mammalian cells: The test item is considered to be non-mutagenic under the conditions of the test.

Endpoint Conclusion:

Justification for classification or non-classification

Three in vitro genetic toxicity studies have been conducted on the test material, an Ames study, a chromosome aberration study and a mouse lymphoma assay. All studies were conducted according to OECD guidelines and GLP and are adequately reported and therefore have been assigned a reliability 1.

The 3 in vitro genetic toxicity studies showed the test material to have no significant effects for gentoxicity. As such, the test material can be considered to be non-classified.