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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was negative in an Ames test, chromosomal aberration study and in an HPRT study.

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

Genetic toxicity in vivo

Description of key information

Micronucleus test in vivo: negative; 2003, OECD Guideline Study.

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

Additional information

Additional information from genetic toxicity in vivo:

Valid experimental data were available to assess the genetic toxicity of the test substance in-vitro and in-vivo.

 

In-vitro:

Gene mutation in bacteria:

In a GLP compliant study according to OECD guideline 471, the 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 TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. The dose range was determined in a preliminary toxicity assay and was set to 50 to 5000 µg/plate in both the range-finding study and the main study, since no visible reduction in the growth of the bacterial background lawn was observed at any dose level. No toxicologically or biologically 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. A small, statistically significant increase in revertant colony frequency was observed in TA1537 (without metabolic activation) at 1500 µg/plate in the range-finding study only. This small increase was considered not to be biologically or toxicologically significant as the response was non-reproducible over two separate experiments, showed no evidence of a dose-response relationship and the revertant colony counts were within the historical maxima for this tester strain. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.

Cytogenicity in mammalian cells:

In a GLP compliant chromosme aberration study following OECD guideline 473), duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Five exposure groups were used: Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system; Experiment 2 included a 24-hour continuous exposure, a 48-hour continuous exposure and a repeat of the 6(18)-hours exposure with metabolic activation. The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 39 to 2500 µg/mL for the 6(18)-hour exposure, both with and without S9, and 78.1 to 1250 µg/mL for the 24 and 48 -hour treatments. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material induced statistically significant increases in the frequency of cells with aberrations in the 6(18)-hour with S9 and 48-hour exposure groups. The test material was shown to be toxic to CHL cells in vitro and near optimal levels of toxicity were achieved in most treatment cases. The test material was shown to be clastogenic to CHL cells in vitro. However, it should be recognised that the clastogenic response was observed at only toxic dose levels and the dose-response relationship was very steep. Furthermore, the response in the presence of metabolic activation was not clearly reproducible.

This study result is questionable due to the following reasons:

There is evidence that the test material or the positive control substances were not correctly applied to the cultures.

1. The low dose was toxic and the high dose proved non-toxic

2. Repeat experiments do not confirm the previous results (e.g. 12 versus 0 exchanges)

3. Replicate dishes show huge differences

4. When effects have been reported they are comparable in size and type with the positive control.

Based on the suspicion of significant technical errors, the above cited study may be flawed and is therefore regarded as invalid. Consequently, a repeat study was performed:

In this second chromosome aberration test performed according to OECD guideline No. 473 and in compliance with GLP, the test item dissolved in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item were close to the range of the solvent control values and within the range of the historical control data. Two significant (p < 0.05) increases were observed in experiment I, in the presence of S9 mix at preparation interval 18 hours after 4 hours treatment with 115.6 and 231.3 µg/ml. Although these increases of 2.5 % and 4.0 % aberrant cells, exclusive gaps, were statistically significant compared to the low response (0.0 % aberrant cells, exclusive gaps) in the solvent control data, the responses are within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps). Therefore, the statistical significances have to be regarded as being biologically irrelevant. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

This negative result supports the invalidity of the initially performed chromosome aberration test. Further support is gained from structural analyses of the test article. Here, no structural alert for clastogenicity or mutagenicity was obtained. Additionally, the test article was also found negative in an in vivo micronucleus test (see below). Therefore, it is concluded that the first chromosome aberration test was invalid and the test article is considered as not clastogenic.

Gene mutation test in mammalian cells:

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, following GLP principled and OECD guideline 476. 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 the results of the present in vitro study, in two experiments performed independently of each other the test substance did not relevantly 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 range of the historical negative control data. The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in our laboratory 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 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 article is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

In vivo:

Micronucleus assay:

The in vivo clastogenic potential of the test substance was evaluated in a micronucleus test according to OECD guideline 474 and in compliance with GLP. Based on a range finding study, the maximum dose level administered was set at 2000 mg/kg and the study was conducted in males only (no sex specific differences were observed in the range finder). Groups of seven mice were treated with 500, 1000 and 2000 mg/kg by intraperitoneal injection. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. 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. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material induced a marked increase in the frequency of micronucleated polychromatic erythrocytes and the test system was therefore considered to be functional. The test material was considered to be non-genotoxic under the conditions of the test.


Justification for selection of genetic toxicity endpoint
Several studies are required for this endpoint, nevertheless the GLP compliant in vivo study was selected here.

Justification for classification or non-classification

Classification, Labelling, 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. The in vitro and in vivo studies are negative for genetic toxicity. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.