Butyl hydrogen maleate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was considered to be non-mutagenic under the conditions of an Ames study.

The test item did not induce any 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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three studies were carried out to assess the test material for genotoxic effects, Summaries as follows:

Ames study

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 (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA were treated with Butyl Hydrogen Maleate 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 was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using a dose range of 5 to 5000 µg/plate, fresh, cultures of the bacterial strains and fresh test item formulations. Additional dose levels (5 and 15 µg/plate) were employed in the second experiment 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 controls 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 lawn in all of the Salmonella strains, with and without metabolic activation (S9-mix) at 5000 µg/plate. There was no visible reduction in the growth of the background lawn of E.coli strain, WP2uvrA. The test item was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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.

Butyl Hydrogen Maleate was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration Test in Human Lymphocytes in vitro

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels dose levels, together with vehicle and positive controls. Four treatment conditions were used for 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.

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...................25, 50, 100, 200, 400, 800

4(20)-hour with S9 (2%)...............50, 100, 200, 400, 800, 1600

24-hour without S9 ......................12.5, 25, 50, 100, 200, 400

4(20)-hour with S9 (1%)...............50, 100, 200, 400, 600, 800

L5178Y TK +/- Mouse Lymphoma Assay

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 (2% 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 in Experiment 1 was 6.72 to 215 μg/ml in the absence of metabolic activation, and 26.88 to 860 μg/ml in the presence of metabolic activation. In Experiment 2 the dose range was 1.69 to 108 μg/ml in the absence of metabolic activation, and 13.5 to 432 μg/ml in the presence of metabolic activation.

Results.

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 study. 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 levels exhibiting acceptable levels of toxicity, either with or without metabolic activation in either the first or the second experiment.

Conclusion.

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Justification for classification or non-classification

Based on the results of the three separate studies, the test material is not considered to be classified for genotoxicity.