Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of an Ames test (OECD 471) it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2uvrA in the absence and presence of S9-mix.

Based on the results of a HPRT test (OECD 476) it is concluded that the test item does not induce gene mutations at the HPRT locus.

Based on the results of an micronucleus test (OECD 487) it is concluded that the test item is not clastogenic or aneugenic in human TK6 ceells.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test with and without metabolic activation) according to OECD guideline 471. The modified Bacterial Reverse Mutation Test according to Prival facilitates azo reduction and is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds.

The tests was performed with 5 strains (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) using a test substance concentration range from 33 to 6200 µg/plate. Precipitation of the test substance was found in the prival preincubation test after the addition of hamster S9 mix from about 3100μg/plate onward.A bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 3100μg/plate onward. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the prival preincubation test without S9 mix or after the addition of a metabolizing system.

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT:

The test substancewas 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). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following doses were tested: 0; 78.1; 156.3; 312.5; 625.0; 1250.0; 2500.0 μg/mL Taking into account the cytotoxicity actually found in the main experiments, the concentration 2500.0μg/mL was not used for evaluation of the1st Experiment with and without S9 mix.Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In this study, in the 1st Experiment in the absence and the presence of metabolic activation the highest concentrations tested for gene mutations were clearly cytotoxic. Due to the determination of a pH shift in the 1st Experiment the pH was adjusted to physiological values in the 2nd Experiment. Then, no cytotoxic effects were obtained at the highest required test substance concentration. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance was not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Micronucleus Assay:

The test substance was assessed for its potential to induce micronuclei in TK6 cells in vitro (clastogenic or aneugenic activity).

Three independent experiments were carried out with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). In the range-finding test for the determination of the experimental doses, no cytotoxicity occurred up to the highest applied concentration 2500 μg/mL, at which no test substance precipitation was observed. Thus, the test substance concentration used in the study ranged from 78.1 to 2500.0 μg/mL. Only the test substance concentrations ranging from 625.0 to 2500 µg/mL were evaluated.A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for TK6 cells. Both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei. In this study, reduced cell count (indicated by relative population doubling) was observed only in the 2nd experiment with metabolic activation at 625 μg/mL. However, the proliferation index (CBPI) was not reduced in any of the test substance concentrations evaluated for micronucleated cells. Thus, a clear cytotoxic effect could not be determined up to the highest tested concentration in any of the performed experiments. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, the test substance was considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.

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 classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.