416-730-1

Endpoint summary

Additional information

Performance and observations

There are reliable, GLP-conform in vitro and in vivo studies available to assess the genotoxic potential of the end product in bacteria, mammalian cells and animals.

The first study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100, TA 1538 and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without S9 mix from aroclor induced rat liver and non-induced Syrian hamster liver. The test item was tested at concentrations of 5000, 2500, 1250, 625 and 312.5 µg/plate.

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation.

 In the second study, test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in cultured human lymphocytes in five independent experiments. The experiments differed in test concentration, exposure duration and metabolic activation. 100 metaphase figures were examined, from each culture, with a maximum of 25 from each slide.

The test article did not cause any significant increases in the proportion of metaphase figures with chromosomal aberrations, in either the presence or absence of S-9 mix. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. Thus, it is concluded that the test item has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

The objective of third in vitro assay was to evaluate the ability of the test item, suspended in water, to induce forward mutations at the thymidine kinase (TK) locus in the mouse lymphoma L5178Y cell line. In the preliminary assay, moderate cytotoxicity was observed at 2500 mug/ml and 5000 mug/ml, however, the precipitate was very heavy and may have contributed to the cytotoxicity. Doses for the definitive mutation assays were selected based on the results of the cytotoxicity assay and the solubility characteristics. Because of the heavy precipitate present at high concentrations of test article, a testing limit of 1000 mug/ml was selected. A single trial of the nonactivation and the S9 metabolic activation mutation assays was performed. Seven treatments from 62.5 mug/ml to 1000 mug/ml were initiated and the six highest doses were cloned for mutant analysis. No cytotoxicity was observed imder either activation condition. None of the six analyzed treatments with or without metabolic activation induced a mutant frequency that exceeded the minimum criterion for a positive response.

Again, the substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three experiments were carried out independently of each other at dose levels of 2.5 - 5200 ug/ml in absence and presence of a metabolic activation syten for 4h and 24h, respectively. Thereafter, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. Precipitation occurred from the lowest applied concentration onward. In all experimental parts in the absence of S9 mix cytotoxicity was observed at strongly precipitating concentrations only. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system. In the 1st Experiment after 4 hours exposure in the absence of S9 mix an increase in the number of mutant colonies was observed in a single culture which was not corroborated in the culture treated in parallel or in the repeat experiment (2nd Experiment).

At last, an in vivo assay was conducted to evaluate the ability of the test article to induce micronuclei in bone marrow polychromatic erythrocytes in mice. Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg bw. In the micronucleus assay, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1250, 2500, and 5000 mg/kg bw. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, euthanized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes.

 

Discussion

The test item did not induce mutations or chromosome aberrations in vitro. The test material was also negative for induction of micronuclei formation in mice. Therefore, the substance is considered as non-mutagenic under the conditions of these tests.

Short description of key information:
An Ames test, mutagenicity assays in mammalian cells, a chromosome aberration test and a micronucleus assay in mice were performed under GLP and according or similar to OECD guidelines 471, 473, 476 and 474, respectively, to evaluate the genotoxic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. The test material was also negative for induction of micronuclei formation in mice. Therefore, the substance is considered as non-mutagenic under the conditions of these tests.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 30th time in Directive 2008/58/EC.

 

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).