Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

In vitro studies

Bacterial reverse mutation assay

In a bacterial reverse mutation assay (CIBA-Geigy No. 78/2566, 1979), conducted similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay but without a marker for cross-linking mutagenicity, e.g. E.coli), the test substance was analysed for mutagenicity by the detection of point mutations in bacteria (histidine-auxotrophic mutants of Salmonella typhimurium). Any mutagenic effects of the test substance is demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were also performed with the addition of an activation mixture (rat liver microsomes and co-factors). The strains TA 98, TA 100, TA 1535, and TA 1537 were treated with the test substance diluted in acetone in doses of 25, 75, 225, 675, and 2025 µg/0.1 ml (plate incorporation test). A negative control and appropriate positive control substances were also evaluated. At concentrations of 225 µg/0.1 ml and higher the test compound precipitated in soft agar. No dose-related biologically relevant increase or doubling in the number of his+ revertants was observed in plate incorporation test in any of the tested strains with or without metabolic activation. Effects on cytotoxicity were not given. According to the results of the present study, the test substance is not mutagenic in the bacterial reverse mutation assay under the experimental conditions chosen. The positive controls yielded the expected results.

 

Chromosomal Aberration Assay

A chromosomal aberration assay (Bozo Research Center Inc. No. M-1281, 2008) conducted according to OECD 473 and under GLP was performed for the purpose of determining the presence or absence of clastogenic activity of the test substance. The chromosomal aberration test was conducted by using cultured mammalian cells (CHL/IU) in the presence and absence of metabolic activation to ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected. Positive controls used were cyclophosphamide with metabolic activation and mitomycin C without metabolic activation. A cell growth inhibition test was initially performed by setting the highest dose at 3600 μg/ml, equivalent to the limit dose of 10 mM. As a result, 50% or more cell growth inhibition was not observed both for the short-term (6 h) and continuous (24 h) treatment methods and thus, the 50% cell growth inhibition concentration (approximate value) was considered at 3600 μg/mL or higher. Only a slight precipitate was noted at all test concentrations. Accordingly, in the chromosomal aberration test, 4 doses each were defined both for the short-term and continuous treatment methods on the basis of the highest dose of 3600 μg/mL by diluting by a common ratio of 2. Increased incidences of structural chromosomal aberrations and polyploidy were not found both in the short-term and continuous treatment methods. Solvent control and positive control values were found to be within range of historical control data.

From these results, the test substance was judged not to have the ability to induce structural chromosomal aberrations and polyploidy under the conditions of the present study.

 

HPRT Test

A HPRT test (Harlan Cytotest Cell Research GmbH No. 50M0860/11 X448, 2012) conducted according to OECD 476 and under GLP was performed to assess the potential of the test item to induce forward gene mutations at the HPRT locus using the Chinese hamster cell line V79. Two parallel cultures were used throughout the assay. The first experiment was performed with a treatment time of 4 h with and without metabolic activation. The second experiment was performed with a treatment time of 24 h without and 4 h treatment with metabolic activation. A range finding pre-experiment was performed using a concentration range of 28.1 to 3600 μg/mL (limit concentration, 10 mM) to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Since no toxicity but precipitation at 225 µg/mL and above with and 450 µg/mL and above without metabolic activation was observed. Concentrations between 14.1 and 900 µg/mL were chosen for the main experiment.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration tested with and without metabolic activation. Solely, in experiment II at 112.5 μg/mL in the second culture with metabolic activation, the induction factor exceeded the threshold of 3.0 (induction factor of 3.6). The isolated effect was biologically irrelevant as it was based on the rather low solvent control of just 7.0 colonies per 1000000 cells and was neither reproduced in the parallel culture I performed under identical experimental conditions, nor in experiment I. Furthermore, the total number of mutant colonies per 1000000 cells at this data point remained well within the range of the historical solvent controls.

Appropriate reference mutagens (ethylmethanesulfonate and 7,12-dimethylbenz(a)anthracene), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion, it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be nonmutagenic in this HPRT assay.


Short description of key information:
In vitro studies:
Similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay; CIBA-Geigy No. 78/2566, 1979): negative
According to OECD Guideline 473 (Chromosomal Aberrations Assay; Bozo Research Center Inc. No. M-1281, 2008): negative
According to OECD Guideline 476 (HPRT Test; Harlan Cytotest Cell Research GmbH No. 50M0860/11 X448, 2012): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vitro data and according to CLP Regulation (EU) 1272/2008 and Directive 67/548/EEC, respectively,

2-(2H-Benzotriazol-2-yl)-4,6-ditertpentylphenol should not be classified for mutagenicity.