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Key value for chemical safety assessment

Additional information

The substance was investigated for its potential to induee gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA (BSL 2009e). The study followed OECD testing guideline 471 and GLP. Experiment II was performed with the modification according to Prival to allow for azo reduction. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, icluding the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 microgrammes/plate. Precipitation of the test item was observed in all tester strains used in experiment l and II (with and without metabolic activation). No toxic effecets of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. No biologically relevant inereases in revertant colony numbers of any of the five tester strains were observed at any concentration level, neither in the presence nor absence of metabolic activation in experiment land II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

The 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 (BASF 2011). The study followed OECD testing guideline 476 and GLP. Two independent experiments were carried out, both with and without the addition of liver S9 mix from 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 and evaluated in this study: 1st Experiment without S9 mix (4-hour exposure period) 0; 10; 20; 40; 80; 1700; 3400 μg/mL and with S9 mix (4-hour exposure period) 0; 10; 20; 40; 80; 1700; 3400 μg/mL; 2nd Experiment without S9 mix (24-hour exposure period) 0; 10; 20; 40; 80; 1700; 3400 μg/mL and with S9 mix (4-hour exposure period) 0; 10; 20; 40; 80; 160; 320 μg/mL. After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. 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, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

Clastogenicity in vitro was determined in a study following OECD testing guideline 473 and GLP (NOTOX 2009d). In the first cytogenetic assay, the substance was tested up to 66 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The substance precipitated in the culture medium at this dose level. In the second cytogenetic assay, the substance was tested up to 100 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix it was tested up to 66 μg/ml for a 3 h exposure time with a 48 h fixation time. The test item precipitated in the culture medium at these dose levels. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Genotoxicity in vivo was determined in the micronucleus assay (OECD 474, GLP) (BASF 2016). This study had to be performed because of non-EU Chemical Regulation testing requirements. It was not performed for the purpose of the EU REACH registration. No genotoxicity was observed at gavage doses of 500, 1000 and 2000 mg/kg bw. According to the results of the present study, there are no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (500 mg/kg, 1000 mg/kg and 2000 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range.

In this study, after single oral administration of the vehicle corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected. Besides the number of cells containing micronuclei in these vehicle control animals was within the range of the historical vehicle control data for PCEs. The administration of the test substance did not influence the erythropoiesis in NMRI mice. Bioavailability of the test substance after oral administration was verified by discolored urine in the dose groups treated with 2000 mg/kg body weight. The positive control substance CPP induced a statistically significant increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data.


Short description of key information:
The substance did not cause gene mutations in an Ames test modified for azo compounds (BASF 2009e) and in the hprt test (BASF 2011). It did not induce chromosome aberrations in cultivated human lymphocytes (NOTOX 2009d). It did not cause genotoxicity in the micronucleus assay in vivo (BASF 2016). All studies were performed according to OECD testing guidelines and GLP.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

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. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221