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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce mutagenicity in bacteria and in mammalian cell culture. It did furthermore not induce micronuclei in human lymphocytes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

AMES

In a GLP-compliant OECD 471 study the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains in a reverse mutation assay. The strains used were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with a dose range of 33 μg - 5000 μg/plate in both standard plate test (SPT) and preincubation test (PIT) each performed with and without metabolic activation (liver S9 mix from induced rats). No bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test up to the highest concentration. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants) was observed using the tester strain TA 1535 and TA 1537 without S9 mix at a concentration of 5000 μg/plate. Test substance precipitation was found from about 333 μg/plate onward in the standard plate test and from about 1000 μg/plate in the preincubation test both with and without S9 mix. The test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). In addition, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. Thus, under the experimental conditions chosen here, it is concluded that the test item is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

HPRT

In a GLP-compliant OECD 476 study the test 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. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). The dose selection of this study was based on the solubility properties of the test substance in an appropriate vehicle and in culture medium in accordance to the recommendations of the current guidelines. Due to lacking cytotoxicity in the pretest concentrations at the border of solubility in culture medium were tested for the occurrence of gene mutations. The test substance did not lead to a biologically relevant increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the 95% control limit of our historical negative control data range. A statistically significant increase in mutant colonies was detected in the 1st Experiment in the absence of metabolic activation at 250.00 μg/mL. The mutant frequency of this test group was clearly within the historical negative control data range. This test group showed strong test substance precipitation in culture medium. Additionally, a statistically significant dose dependent increase in mutant colonies was observed in this experimental part. This observation was solely based on the high value at the highest applied concentration. All further exposed test groups led to corrected mutation rates between 2.51 and 3.69 colonies per 106 cells. Nevertheless, the values obtained for the corrected mutation frequency of this experimental part were within the range of the 95% control limit (MFcorr.: 0.00 – 7.19 per 106 cells). Therefore, these findings have to be considered as artifactual and, thus, these observations have been regarded as biologically irrelevant. Additionally, no statistically significant dose-dependent increase in mutant colonies was observed in the other experimental parts of this study after 4 hours treatment either in the absence or presence of metabolic activation. The mutation frequencies of the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, in the absence and the presence of metabolic activation, the test item is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

MNT

The test item suspended in Ethanol was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group, two parallel cultures were analyzed. 1000 binucleate cells per culture were evaluated for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 300 µg/mL was chosen with regard to the solubility properties and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. In Experiment I, precipitation of the test item in the culture medium was observed at 25.0 µg/mL and above in the absence of S9 mix and at 100 µg/mL and above in the presence of S9 mix at the end of treatment. In addition, precipitation occurred in Experiment II in the absence of S9 mix at 25.0 µg/mL and above at the end of treatment. No relevant influence on osmolarity or pH was observed. No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed, in either experimental part, up to the highest applied concentration. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. Demecolcine (75 ng/mL), MMC (0.8 µg/mL) or CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.