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Bacterial reverse mutation assay (Ames test):

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with suspensions of 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 was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, 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 and without metabolic activation.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 ug/plate. A precipitate was observed at and above 1500 ug/plate, this 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 mammalian chromosome aberration tests:

Results from five chromosome aberration studies are available, both conducted on the test material and on three closely related structural analogues.

Studies on test material: Results are available from two chromosome aberration studies (one using human lymphocytes and one using Chinese Hamster lung cells (CHL)).

In the studies using lymphocytes, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

In a test conducted to a Japanese method using CHL cells, which does not conform to EC/OECD requirements, the test substance induced numerical chromosomal aberrations dose-dependently within the dose range of 125 – 500 µg/ml in the 48 h treatment by the direct method, and 500 – 5000 µg/ml in the group without S9 mix by the metabolic activation method, respectively. No structural aberrations were noted in this study.

The positive controls (MMC and CPA) induced evident structural chromosomal aberrations.

Studies on structural analogues: Results have been read-across from chromosome aberrations studies using CHL cells from three structurally related analogue substances.

The three analogue substances were considered to be non-clastogenic to CHL cells in vitro.

Mammalian cell gene mutation assay (mouse lymphoma assay):

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 Guidelines for Testing of Chemicals NO.476"In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

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 six dose levels, in duplicate, together with vehicle (solvent) 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 six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9final 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 and for Experiment 1 and 2 was 64.77 to 1036.25 µg/ml in both the absence and presence of metabolic activation.

The maximum dose level used in the Mutagenicity Test was limited by the formulation of the test item and the maximum achievable dose level was 1036.25 µg/ml. Precipitate of the test item was observed at and above 64.77 µg/ml in the Mutagenicity Test.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

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.


Justification for selection of genetic toxicity endpoint
All available study data has been reviewed to assess the genetic toxicity / mutagenicity of the test material.

Short description of key information:
Negative results for mutagenicity were obtained in the in-vitro studies mutagenicity studies conducted on the test material and three surrogate read-across substances. Studies included:
- Bacterial reverse mutation assay (Ames test)
- In vitro mammalian chromosome aberration test (CHL cells and human lymphocytes)
- mammalian cell gene mutation assay (mouse lymphoma assay)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was found to be:

- non-mutagenic in a bacterial reverse mutation assay (Ames test).

- non-mutgenic to L5178Y cells in a mouse lymphoma assay.

- non-clastogenic in in-vitro chromosome aberration studies (in CHL cells and human lymphocytes).

In a test conducted to a Japanese method using CHL cells the test substance induced numerical chromosomal aberrations but no structural aberrations were noted in this study and the result is considered to be negative. It has been assessed that the numerical aberrations, which was noted at toxic dose levels, was most likely to be a cytotoxic drive response on the mitotic apparatus and as no structural aberration response was observed, not to be an indication of a true clastogenic effect.

Three structurally similar analogue substances were found to be non-clastogenic in in-vitro chromosome aberration studies.

Based on the above, the substance has been assessed as not requiring classification as a germ cell mutagen.