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Additional information

There is data from in vitro assays available addressing gene mutation in bacteria, clastogenic activity in mammalian cells and gene mutation in mammalian cells. Gene mutation in bacteria was assessed by the Reverse Mutation Assay (Ames test) conducted with 5 Salmonella typhimurium strains (TA1535, TA1537, TA1538, TA98 and TA 100) and the yeast strain Saccharomyces cerevisiae D4, which was comparable to OECD guideline 471 (Jagannath, 1978). All assays were conducted in presence and absence of a metabolic activation system consisting of Aroclor1254-induced rat liver S9 with test substance concentrations ranging from 0.1 to 500 µg/plate; additionally 1000 µg/plate was included in a repetition test with TA98. Although there was no strain included to detect cross-linking mutagens according to the actual version of the guideline, the study was acceptable according to the standards of the time of conduction. In this study no mutagenic effects on bacteria or yeast were observed with or without metabolic activation at any of the concentrations tested.

The clastogenic activity of the test substance was investigated by a Chromosomal Aberration Assay conducted with a Chinese Hamster lung cell line according to OECD guideline 473 (Wright, 2006). Duplicate cell cultures were exposed for either 6 hours to concentrations up to 156.25 µg/mL in presence or absence of a metabolic activation system consisting of Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix, followed by an 18-hour recovery period, or for 24 hours to concentrations up to 78.13 µg/mL in the absence of metabolic activation. Three concentrations and the vehicle control were chosen for analysis of 200 metaphases; the highest concentration chosen was the lowest one at which precipitation occurred. The chromosomes were analysed for structural aberrations comprising chromosome and chromatid breaks and exchanges, gaps, numerical aberrations and multiple aberrations. Under the conditions of this study the test substance did not induce any statistically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in presence or absence of a metabolic activation system after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.

Mutagenicity of the test substance in mammalian cells in vitro was determined in the L5178Y mouse lymphoma cell line according to OECD guideline 476 (Verspeek-Rip, 2010). The number of mutants derived from 5 exposure plates was determined in 2 independent exeriments. In the first experiment the mouse lymphoma cells were exposed for 3 hours to test substance concentrations ranging from 0 to 1.0 µg/mL in the absence and presence of a metabolic activation system consisting of 8% (v/v) Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix. In this setup precipitation occurred at concentrations ≥ 0.6 µg/mL, no cytotoxicity was observed, and there was no significant increase of mutant frequencies at the TK locus compared to the control groups. These findings were confirmed by a second independent experiment conducted with the same test substance concentrations, but with 3-hour exposure in the presence of 12% (v/v) rat liver S9-mix or 24-hour exposure without metabolic activation. In conclusion the test material did not induce gene mutations in mammalian cells in vitro.

According to Regulation (EC) No 1907/2006, Annex IX, 8.4., column 2, testing for genetic toxicity in vivo is not indicated as the test substance did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
In vitro:
Gene mutation in bacteria (Reverse Mutation Test, OECD 471): negative
Cytogenicity in mammalian cells (Chromosomal Aberration, OECD 473): negative
Gene mutation in mammalian cells (Mouse Lymphoma Assay, OECD 476): negative

In vivo:
Data waiving - No in vivo testing required as none of the in vitro tests were positive for genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.