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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation: OECD 471: negative

Gene mutation in mammalian cells: OECD 476: negative

Cytogenicity in mammalian cells: OECD 473: negative

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

Genetic toxicity in vivo

Description of key information

Micronucleus test: OECD 474: negative

UDS test: OECD 486: negative

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

Additional information

There are reliable in vitro and in vivo genotoxicity studies available.

In vitro:

- Gene mutation in bacteria:

A GLP guideline study according to OECD guideline 471 is available with the test material (2004). Salmonella typhimurium strains TAI535, TAI 537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at five dose levels up to 5000 µg/plate, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.

- Gene mutation in mammalian cells:

A GLP guideline study performed according to OECD guideline 476 is available with the test material (2012). The study was performed to investigate the potential of the test material to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest applied concentration of 2800 μg/mL was limited by the solubility properties of the test item in DMSO and aqueous medium.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the two main experiments.

Appropriate reference mutagens, 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 material did not induce gene mutation at the HPRT locus in V79 cells and thus is considered to be non-mutagenic in this HPRT test.

- Cytogenicity in mammalian cells:

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test was performed according to OECD Guideline 473 and GLP (2005). Duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test material together with vehicle (DMSO) and positive controls. Three treatment regimens were used: a 6 (18)-h exposure, both with and without metabolic activation (S9) and a 24-h continuous exposure in the absence of S9. The dose levels used were selected on the basis of molecular weight, solubility and the results of a preliminary toxicity test. The dose range for the 6 (18)-h exposure groups, both with and without S9, and the 24-h continuous exposure group was 276.9 to 4430 µg/mL. 100 metaphases per dose were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.

Precipitation was observed at 2215 μg/mL and above in the presence and absence of S9 mix. The vehicle control had frequencies of cells with aberrations within the range expected for the CHL cell line. All of the positive control materials induced highly significant increases in the frequency of cells with aberrations demonstrating the validity of the test. The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in any treatment group with or without metabolic activation. The dose levels of the test material induced optimum levels of toxicity to CHL cells in the 24-h continuous exposure group, but only modest levels of toxicity in both of the 6 (18)-h exposure groups at dose levels up to the limit dose.

In conclusion, the test substance did not induce structural chromosomal aberrations in CHL cells in vitro in the presence and absence of metabolic activation under the experimental conditions reported. Therefore, the test substance is considered to be not clastogenic in this chromosome aberration test, when tested up to precipitating or the highest evaluable concentrations.

In vivo:

- Cytogenicity study:

A GLP guideline study performed according to OECD guideline 474 (Micronucleus Test) is available with the test material (2005). The study was performed to assess the potential of the test material to produce damage to chromosome or aneuploidy when administered to mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1000 mg/kg and with 500 and 250 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48 -hour test material groups when compared to their concurrent vehicle control groups. However, there was a modest reduction in PCE/NCE ratio observed with the test material in the 48-hour 1000 mg/kg dose group when compared to the concurrent control, and this accompanied by the presence of clinical signs was taken to indicate that systemic absorption had occurred.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

In conclusion, the test material was considered to be non-genotoxic under the conditions of the test.

- DNA damage/repair:

A GLP guideline study performed according to OECD guideline 486 (Unscheduled DNA Synthesis Test with Mammalian Liver Cells in Vivo) is available with the test material (2012). The test material was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The test item was dissolved in 30% DMSO and 70% PEG 400, which was used as vehicle control. After a single oral treatment and a post-treatment period of 4 and 16 hours, respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR (methyl-3H-thymidine), which is incorporated if UDS occurs. The test item was tested at dose levels of 1000 and 2000 mg/kg bw and 4 and 16 hours preparation intervals. Hepatocytes of four males and four females for each experimental group including the controls were assessed for the occurrence of UDS. In the negative control group of the 4 h preparation interval, due to failed perfusion, only three males were assessed for the occurrence of UDS, and in the low and high dose group of the 16 h preparation interval, only three males were treated by mistake. For the positive control group of the 16 h preparation interval four males were evaluated but only three males are reported, since the cell viability of the hepatocytes isolated from one male animal of the 2-AAF (2 -acetylaminofluorene) positive control group was low (57%).

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test item. No clinical signs of toxicity were observed in any treated males or females. None of the tested dose levels revealed UDS induction in the hepatocytes of the treated animals as compared to the corresponding vehicle controls. Appropriate reference mutagens [DMH (N,N'-dimethylhydrazinedihydrochloride), 80 mg/kg bw and 2-AAF, 100 mg/kg bw] were used as positive controls. Treatment with the positive control substances revealed distinct increases in the number of nuclear and net grain counts.

In conclusion, it can be stated that under the experimental conditions reported, i.e. oral administration up to 2000 mg/kg bw, the maximal tolerated dose, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the test material is considered to be non-genotoxic in this in vivo UDS test system.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.