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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria (Ames):

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method at five 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 range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.

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.

The test material was considered to be non-mutagenic under the conditions of this test.

In-vitro cytogenicity in mammalian cells (chromose aberration): Read-across from structural analogue (EC 700 -043-1)

The report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. I

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Three treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2 in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that was limited by the lowest precipitating dose level.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

In-vitro gene mutation in mammalian cells (Mouse Lymphoma Assay): Read-across from structural analogue (EC 700 -043-1)

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls. The exposure groups used were as follows: 4-hour exposures with and without metabolic activation and 24 hours without metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 39.06 to 625 μg/ml for all three of the exposure groups.

The maximum dose level used in the mutagenicity test was limited to 625 μg/ml. This was due to the onset of a greasy / oily precipitate effectively reducing the exposure of the test material to the cells in the preliminary toxicity test. Whilst precipitate was observed at the end of the exposure periods of the mutagenicity test, it was not carried over into the maintenance phases of the test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant or dose-related increases in the mutant frequency at any dose level, in any of the three exposure groups.

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three in vitro mutagenticity studies are available to assess the genetic toxicity of the substance.

Bacterial reverse mutation assay (Ames test, OECD 471): The test material was considered to be non-mutagenic under the conditions of this test.

In vitro mammalian chromosome aberration test (OECD 473) (conducted on structural analogue): The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mammalian cell gene mutation assay (mouse lymphoma assay, OECD 476) (conducted on structural analogue: The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for classification or non-classification

Based on negative results in in-vitro studies conducted on the substance and a structural analogue, the substance does not need to be classified for germ cell mutagenicity.