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Key value for chemical safety assessment

Additional information

Ames Test

The mutagenic potential of the test substance was investigated in an Ames test according to OECD guideline 471, performed with Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli strain WP2 uvrA in the presence and absence of a metabolic activation system (S9 mix). Based on the results in the dose finding test, the maximum tested concentration in the main test was 5000 µg/plate with and without S9-mix. None of the tested concentrations led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the negative control either with or without metabolic activation. Normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria. Therefore, based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used (Ciba-Geigy, 1992).

Chromosome Aberration

The clastogenic potential of the test article was evaluated in a GLP-compliant chromosome aberration study following OECD guideline 473 using V79 cells in vitro. According to the results of this study, the test substance led to a statistically significant and biologically relevant increase in the number of structural chromosomal aberrations excl. gaps without S9 mix in two experiments (2nd and 3rd Experiment) performed independently of each other after 18 hours exposure time at prolonged 28 hours sampling time. In all further experimental parts performed in this study after 4 and 18 hours treatment at 18 hours sampling time in the absence and/or presence of metabolic activation no biologically relevant increase in the number of aberrant metaphase cells was observed. The types and frequencies of structural chromosome aberrations of these experimental parts were within the range of the concurrent vehicle control values and clearly within in the range of the historical negative control data. In the 1st Experiment after 4 hours treatment at 18 hours sampling time without S9 mix, due to inhomogeneous data an increased sample of 400 metaphase cells was scored in the negative control group and at 2000 μg/mL each. Similarly, at 2000 μg/mL in the 1st Experiment with S9 mix an increased sample of 400 metaphase cells was scored. However, in the 2nd Experiment after 18 hours exposure at prolonged 28 hours sampling time a weak, statistically significant chromosome-damaging effect (8.5% aberrant metaphase cells, excl. gaps) slightly exceeding the historical negative control data range (0.0 – 5.5% aberrant metaphase cells, excl. gaps) was observed at the highest scorable concentration (500 μg/mL). This test group showed a clear cytotoxic effect indicated by reduced cell numbers of 48.6% of control. The 3rd Experiment was performed to corroborate this finding. For this purpose a concentration range from 300 to 700 μg/mL was used. The highest applied concentration showed no clear cytotoxicity. At 700 μg/mL the number of aberrant metaphases was statistically significant increased (8.5%) compared to the concurrent vehicle control group (2.5%). The aberration rate of this test group slightly exceeded the historical negative control range (0.0% - 5.5% aberrant metaphases, excl. gaps). Therefore, the slight increase of structural chromosome aberrations, confirmed in a repeat experiment, has to be regarded as biologically relevant. No relevant increase in the number of cells containing numerical chromosomal aberrations was observed in the absence and the presence of metabolic activation The structural chromosome aberration rates 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 structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system 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, under the experimental conditions chosen here, the conclusion is drawn that the test article is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence of metabolic activation after 18 hours exposure at prolonged preparation interval (BASF, 2010).

HPRT

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. According to the results of this study, the test substance did not lead to a biologically relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other. The mutant frequencies at any concentration were within the range of the concurrent negative control values and within the range of the historical negative control data. A single increase in the number of mutant colonies exceeding the recent negative control data base for the 24 hours exposure interval was observed at 1000 μg/mL in the 3rd Experiment in the absence of metabolic activation. The statistical evaluation did not show any linear dose-response relationship and the value was within the historical negative control data range including all modifications. Therefore, this finding has to be regarded as biologically irrelevant. The mutation frequencies of the vehicle control groups were within the historical negative control data range including all vehicles used in the 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 MCA 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, under the experimental conditions chosen here, the conclusion is drawn that the test item is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation (BASF, 2010).

In vivo micronucleus test

No data is available for the test item. Therefore, study data obtained with a structurally related compound is used to address this data gap in a read-across approach. For read-across justification see the Annex of the CSR. Similarly to the test item, the structurally related compound was also positive in a chromosome aberration study. Therefore a micronucleus assay was performed (Mitsui Chemical Inc., 2001). This assay with bone marrow cells of rats was performed in accordance with die OECD TG 474. The read-across compound was administered twice orally (during a 24 hour interval) to male Crj:CD (SD) IGS rats at 250, 500, 1000 and 1500 mg/kg. The incidence of micronucleated immature erythrocytes was evaluated in rat bone marrow 22 to 24 hours after the final administration. Distilled water was used as a negative control, and cyclophosphamide monohydrate (CP) 20 mg/kg as a positive control. The result of this assay was as follows. 1) No significant increase was noted in the frequency of micronucleated immature erythrocytes in any test article group, in comparison with the negative control group. A significant low value was noted in the immature erythrocyte ratio of all doses compared to the negative control group. 1 of 6 animals died at 1500 mg/kg. 2) A significant difference was noted in the frequency of micronucleated immature erythrocytes and in the ratio of immature erythrocytes in the positive control group compared to the negative control group. 3) After exposure of the test article to bone manow erythrocytes in rats, it was confirmed that a significant decrease in the immature erythrocyte ratio occurred at all doses compared to the negative control group. Therefore, it was concluded from the results described above, that under the conditions of this study, this substance did not induce clastogenic activity in the bone marrow erythrocytes of rats.


Justification for selection of genetic toxicity endpoint
The selected study represents the whole battery of tests, all of which were performed according to current guidelines.

Short description of key information:
The test aticle was not mutagenic in an Ames test an in an in vitro gene mutation assay in mammalian cells. It was clastogenic in vitro in CHO cells in the absence of a metabolizing system but a structurally related compound was negative in vivo, therefore there is also no concern for clastogenicity.

Endpoint Conclusion: No adverse effect observed (negative)

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 1272/2008 (CLP). As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the sixth time in Regulation (EC) No 605/2014.

Dangerous Substance Directive (67/548/EEC)

The available study are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.