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Bacterial Reverse Gene Mutation Assay - The Ames Test

Introduction.

The object of the study was to assess the mutagenic potential of the test material in a bacterial system, following a test method based on OECD Guideline No. 471: Genetic Toxicology: Salmonella typhiumurium, Reverse Mutation Assay.

Methods.

Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were used in the study.

The dose levels used were 5000, 500, 50 and 5 µg/plate in a dose range finding test, and 5000, 1500, 500, 150 and 50 µg/plate in the subsequent mutation tests.

Each dose level was tested in triplicate, both with and without metabolic activation (S9 mix).

Results.

In the dose range finding test, DIXT was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with DIXT at any dose level, either in the presence or absence of metabolic activation (S9 mix).

Conclusion.

It is concluded that no evidence of mutagenic potential of DIXT was obtained in this bacterial test system at the dose levels used.

Chromosome Aberration Test

DIXT was tested in vitro to determine whether it could cause chromosomal aberrations in a mammalian cell line derived from Chinese hamster ovary tissue. The cells were routinely grown and subcultured in tissue culture medium at 37°C in a humid atmosphere containing 5% carbon dioxide. They were incubated with the test compound both with and without supplementary metabolic activation (rat S9 mix).

A preliminary toxicity test was carried out to assess the effect of the compound on the mitotic index of cultured CHO cells. The results of this test indicated that a top dose level of 3 µg/ml should be used for the metaphase analysis in the absence of metabolic activation and 20 µg/ml in its presence.

In both the absence and presence of metabolic activation, DIXT caused no statistically significant increases in the level of chromosomal aberrations at any dose level, when compared with the solvent control.

Both positive control compounds showed statistically highly significant increases in the number of chromosomal aberrations when compared with solvent controls. This demonstrates the sensitivity of this test system and the efficacy of the S9 mix.

It is concluded that DIXT has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.


Short description of key information:
In-vitro studies were conducted on the substance:
- Reverse mutation assay 'Ames Test' using S. typhimurium
- Chromosome aberration test in Chinese hamster ovary (CHO) cells
The studies gave negative results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on negative results in the following in-vitro studies, the substance is not classified for mutagencity.

 

- Reverse mutation assay 'Ames Test' using S. typhimurium:

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

- Chromosome aberration test in CHO cells:

The test item is considered to be non-clastogenic in this chromosome aberration test.