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OECD Guideline 471: Bacterial Reverse Mutation Test:

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to seven 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 ranged between 15 and 5000µg/plate,depending on bacterial strain type. The experiment was repeated on a separate day (pre-incubation method) using an amended dose range (5 to 5000 µg/plate), fresh cultures of the bacterial strains and fresh test item formulations.

 

Additional dose levels were included and an expanded dose range was selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

Results

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 item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies of TA100, TA1535 and TA1537 (absence of S9-mix only), initially from 1500µg/plate.Weakened lawns were also noted for TA100 (presence of S9-mix) at 5000µg/plate using the plate incorporation modification and TA98 (absence of S9-mix) at the same dose level employing pre-incubation methodology. No toxicity either as weakened bacterial background lawns or reductions in colony frequency were noted for any of the remaining tester strains. The test item was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (greasy in appearance) was noted at 5000 µg/plate, this observation 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 item, either with or without metabolic activation or exposure method.

Conclusion

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

OECD Guideline 473: Chromosome Aberration Test:

Methods. 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, a 4-hour exposure 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-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour 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.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Group

Final Concentration of Test Item (µg/ml)

4(20)-hour without S9

20, 40, 80, 160, 320, 480

4(20)-hour with S9 (2%)

40, 80, 160, 320, 480, 640

24-hour without S9

40, 80, 160, 240, 320, 480

4(20)-hour with S9 (1%)

40, 80, 160, 320, 480, 640

 

 

Results.

All vehicle (solvent) control groups 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 indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item 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 included a dose level that induced or exceeded approximately 50% mitotic inhibition.

 

Conclusion.

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

OECD 476: CHO HPRT forward mutation assay:

Introduction

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Methods

Chinese hamster ovary (CHO) cells were treated with the test item at up to eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls. Four treatment conditions were used for the test, i.e. in Experiment 1, a 4-hour exposure in

the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9

concentration); whilst in the absence of metabolic activation the exposure was time was extended to 24-hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group

Final concentration of the test item (µg/ml)

4-hour without S9

18.75, 37.5, 75, 150, 300, 450, 600, 800

4-hour with S9 (2%)

9.38, 18.75, 37.5, 75, 150, 300, 600, 800

24-hour without S9

2.5, 5, 10, 20, 40, 50, 60, 80

4-hour with S9 (1%)

10, 20, 40, 80, 160, 320, 640

Results.

The vehicle (DMSO) controls gave mutant frequencies within the range expected for CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

An increase in mutant frequency of greater than 20 x 10-6was seen in the 4-hour exposure group in the absence of S9 in Experiment 1 at the maximum dose plated for mutant frequency (450 μg/ml). However, this response was considered to be related to the toxicity seen at this dose level and it was only marginally above the acceptable range and was part of an experiment where the background (spontaneous) mutant frequency of the vehicle controls was high. There was no response seen in the 24-hour exposure group in the absence of S9 in Experiment 2 and therefore the response seen in Experiment 1 was considered to be of no toxicological significance. An increase in the mutant frequency of greater than 20 x 10-6 in the 4-hour exposure group with metabolic activation of Experiment 2 at 80 μg/ml was also considered to be of no toxicological significance as it was only marginally above the upper limit of the historical range for a vehicle control, at a dose level at the limit of acceptable toxicity and was set against a low vehicle control value. There was no evidence of a response in the 4-hour exposure group in the presence of S9 in Experiment 1 and this also provided evidence that the response in Experiment 2 was of no biological relevance.

Conclusion.

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.



Justification for selection of genetic toxicity endpoint
Three separate in vitro genetic toxicity studies have been conducted on the test item as follows:

OECD Guideline 471 Bacterial Reverse Mutation Test
OECD Guideline 473 Genetic Toxicology: Chromosome Aberration Test
OECD 476 In Vitro Mammalian Cell Gene Mutation Tests

All 3 studies have been conducted according to OECD Guidelines and GLP and are adequately reported. All studies have been assigned a reliability 1.

Short description of key information:
OECD Guideline 471 Bacterial Reverse Mutation Test:
The test item was considered to be non-mutagenic under the conditions of this test.

OECD Guideline 473 Chromosome Aberration Test:
The test item did not induce statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

OECD 476 In Vitro Mammalian Cell Gene Mutation Tests:
The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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

 

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

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

- Chromosome aberration test in human lymphocytes:

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

- CHO HPRT forward mutation assay:

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

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