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In vitro gene mutation study in bacteria (Ames test):

In the key study (Bowles & Thompson 2010) Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and Escherichia colistrain WP2uvrA-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at five 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 the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. The test material was considered to be non-mutagenic under the conditions of the test.

In vitro cytogenicity study in mammalian cells (chromosome aberration test):

In the key study (Lacey 2010) 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 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 test material exhibited moderate toxicity and 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 was limited by the presence of heavy precipitate. The test material was considered to be non-clastogenic to human lymphocytesin vitro.

In vitro gene mutation study in mammalian cells (mouse lymphoma assay):

In the key study (Flanders 2010) 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 material at six 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 material at six 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 material was selected following the results of a Preliminary Toxicity Test. The test material dose range for both Experiment 1 and Experiment 2 was 156.25 to 5000 µg/ml in both the absence and presence of metabolic activation. Precipitate of test material was observed at all of 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 materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material 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 using a dose range that included the maximum recommended dose level of 5000 µg/ml.


Short description of key information:
Bentonite acid leached was tested for genotoxicity in an Ames (OECD TG 471) , chromosome aberration (OECD TG 473) and a mouse lymphoma assay (OECD TG 476). Results were negative in all three tests.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Bentonite acid leached was negative in all three genotoxicity assays and therefore does not warrant a classification in accordance to CLP or DSD.

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