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Bacterial Mutation Assay

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TAl535, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co—factors). The dose range for the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of the range-finding test, and was 1.5 to 5000 µg/plate.

Eight test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.

Results

The vehicle (DMSO) 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 maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix although substantial reductions in revertant colony frequency were observed to several tester strains (particularly TA1535 and TA1537) at the upper dose levels. In the main test reductions in revertant colony frequency were again noted at the upper dose levels for several of the Salmonella strains. However, in this experiment a weakening of the bacterial background lawns was noted at 5000 ug/plate for all of the Salmonella strains dosed in the absence of S9-mix and to TA1535 and TA1537 in the presence of S9-mix. Escherichia coli strain WP2uvrA showed no evidence of toxicity either as weakened bacterial background lawns or reductions colony frequency in either experiment. The sensitivity of the bacterial tested strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9 -mix. A test item film (cream coloured in appearance) was observed at and above 1500 ug/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, 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 S9-mix in the second mutation test.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirmi ng the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusion

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

Mammalian Cell Chromosome Aberration Test

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. A main experiment was performed using three exposure conditions; a 4-hour exposure in the absence and presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period, and a 24 hour exposure in the absence of metabolic activation. The dose levels used in the main experiments were selected using data from the preliminary toxicity test.

Results

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method operating as expected. The test item did demonstrate marked toxicity in all three exposure groups of the preliminary toxicity test, however, the test item also formed precipitate in the upper half of the dose range of the prelimianry toxicity test and therefore the dose range for the main experiment was limited to the lowest precipitating dose level. The test did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a dose level that was the lowest precipitating dose level in the exposure groups of the main experiment.

Conclusion

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

Mammalian Cell Gene Mutation Assay

Introduction

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 in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

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). In Experiment 2, the cells were treated with the test item at ten dose levels using a 4-hour exposure group in the presence of metabolic activation (2% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose level used in the main test was limited by test item induced toxicity.

Results

A precipitate of test item was not observed at any dose level. The vehicle (DMSO) 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 metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.


Justification for selection of genetic toxicity endpoint
The bacterial mutation assay (Ames test), chromosome aberration test and mammalian cell mutation test were all negative (non-genotoxic).

Short description of key information:
A Klimisch grade 1, GLP compliant bacterial mutation assay performed according to the OECD 471 test method.
A Klimisch grade 1, GLP-compliant mammalian cell chromosome aberration test performed according to the OECD 473 test method.
A Klimisch grade 1, GLP-compliant mammalian cell gene mutation assay performed according to the OECD 476 test method.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification for mutagenicity is proposed according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).