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Ames

In the key study performed according to OECD Guideline 471 the test item was evaluated in the Ames/Salmonella-E.coli liquid Pre-incubation assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of Salmonella (TA1535, TA1537, TA1538, TA98 and TA100) and at the tryptophan locus in one Escherichia coli strain (WP2 uvrA), in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of the test material was initially evaluated in a prescreen by treating duplicate cultures of strains TA1538, TA100 and WP2 uvrA with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9. Results of the pre-screen indicated the test item produced inhibited growth (characterised by a reduced background lawn and/or the presence of pindot colonies) or complete toxicity in all three tester strains at all doses evaluated. In addition, the test article was incompletely soluble at dose >= 500 ug/plate. Therefore, the test item was re-evaluated under identical conditions at doses of 0.5, 1.67, 5.0, 16.7, 50 and 167 ug/plate. OS #43247H again produced inhibited growth or complete toxicity in strains TA1538 and TA100 at dose >= 5.0 ug/plate, and in strain WP2 uvrA at doses >= 16.7 ug/plate.

Based upon these findings, the test material was evaluated in triplicate cultures in all five Salmonella strains at doses of 0.05, 0.167, 0.5, 1.67, 5.0, 16.7 ug/plate, and in strain WP2 uvrA at doses of 0.167, 0.5, 0.67, 5.0, 16.7 and 50 ug/plate, in the presence and absence of S9. A further confirmatory test using modified dose ranges was performed both in the presence and absence of S9 and a third test was performed with all strains in the presence of S9 only.

Toxicity was observed that varied both between strains and between the exposure conditions in the presence and absence of S9. However, overall a satisfactory number of analysable dose levels was achieved in most cases, both in the presence and absence of S9. There were no statistically significant and dose-related increases in the frequency of revertant colonies seen for any strain of bacteria, either in the presence or absence of S9.

Chromosome aberration

The method used in this study followed that described in OECD Guidelines for Testing Methods (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EEC. The study design also meets the requirements of the UK Dept. of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used in 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, the 4 hours 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 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 toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two seperate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

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

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. Thethod 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 eight 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). 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 (1% S9) 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 was 0.63 to 25 µg/ml in the absence of metabolic activation, and 1.25 to 30 µg/ml in the presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 1.25 to 30 µg/ml in the absence of metabolic activation, and 0.63 to 25 µg/ml in the presence of metabolic activation.

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of test item was not observed at any of the dose levels in the Mutagenicity 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 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 the 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:
The test material is negative in vitro in the absence and presence of S9.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There are no indications that the substance is mutagenic or clastogenic in vitro with or without metabolic activation.