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EC number: 406-750-9 | CAS number: 129757-67-1
The test article was negative in an Ames, a chromosomal aberration and in a mammalian cell gene mutation assays. All studies were guideline and GLP compliant.
SUMMARY OF RESULTS
* Microscopically or macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
s Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)
n.c.1 Culture was not continued since a minimum of only four analysable concentrations is required
n.c.2 Culture was not continued since only one concentration beyond the solubility limit is required
n.d. Not determined
1 Aceton 1% (v/v) 2 EMS 400 μg/mL 3 DMBA 1.25 μg/mL
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. One experiment with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation) was carried out. According to strong precipitation (from 19.5 μg/mL onward) of the test substance in culture medium in an initial range-finding cytotoxicity test, the maximum concentration to be used in this study was determined to be 40.0 μg/mL. The following concentrations were tested (test groups printed in bold type were evaluated for gene mutations):
1st Experiment, without S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 7.50; 10.00; 15.00; 20.00; 40.00 μg/mL
1st Experiment, with S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 7.50; 10.00; 15.00; 20.00; 40.00 μg/mL
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. The highest tested concentrations in the main experiment showed clear test substance precipitates in culture medium macroscopically at the end of exposure period. No cytotoxicity was observed up to the highest concentrations evaluated for gene mutations. 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. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Cytogenicity in vivo: micronucleus test, mouse: negative (GLP, OECD 474, 1990)
Gene mutation in bacteria:
In a GLP conform 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 investigations were performed without and with (Aroclor 1254-induced rat liver S-9 mix) microsomal activation with the following concentrations of the trial substance: 313, 625, 1250, 2500 and 5000 µg/plate. In order to confirm the results, the experiments were repeated. In the experiments performed without and with microsomal activation, none of the tested concentrations of the test substance led to an increase in the incidence of both histidine- or tryptophan-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and E. coli used in these experiments
According to the results of the present study, the test substance is not a mutagenic agent in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Cytogenicity in mammalian cells:
In a GLP conform study according to OECD guideline 473, the substance was assessed in CHO-K1 cells in vitro for possible clastogenic activity both in the presence and in the absence of a metabolizing system. In the original study, the concentrations of 150.0, 300.0 and 600.0 µg/ml were selected for chromosome analysis from the first experiment (18 hours treatment without activation) and from the second experiment (3 hours treatment with activation and 15 hours recovery time) each. In the first and the second experiment of the confirmatory study, the same concentrations as mentioned above in the original study were selected for chromosome analysis. The concentrations of 150.0, 300.0 and 600.0 /ig/ml were selected for chromosome analysis in the third experiment (42 hours treatment without activation) and in the fourth experiment (3 hours treatment with activation and 39 hours recovery time) each. Two hundred metaphases were examined from the vehicle control and from the cultures treated with the various concentrations of the test substance. At least fifty metaphases each from the appropriate positive controls were analyzed. In the original study, in the first experiment (18 hours treatment, 0 hours recovery) performed without microsomal activation and in the second experiment (3 hours treatment, 15 hours recovery) performed with microsomal activation, the number of cells with specific chromosomal aberrations in the treatment groups showed no marked difference in comparison with the negative control. In the confirmatory study, in the first experiment (18 hours treatment, 0 hours recovery) performed without microsomal activation, in the third experiment (42 hours treatment, 0 hours recovery) performed without microsomal activation as well as in the fourth experiment (3 hours treatment, 39 hours recovery) performed with microsomal activation, the number of cells with specific chromosomal aberrations in all treatment groups showed no marked increase in comparison with the negative controls. In the second experiment (3 hours treatment, 15 hours recovery) performed with microsomal activation, a weak increase in the number of cells with specific chromosomal aberrations (mainly due to chromatid breaks) was registered. However, these changes observed are within the range of spontaneous aberrations occasionally seen in control cultures (historical control data).
It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in vitro treated with the test substance.
Mammalian cell gene muation assay:
In a GLP-cpmpliant HPRT assay following OECD guideline 476, the test article did not lead to a biologically relevant or dose-dependent increase in the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were within the 95% control limit of our historical negative control data. Additionally, no statistically significant dose-dependent increase in mutant colonies was observed after 4 hours treatment either in the absence or presence of metabolic activation. The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data. 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/or 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 item is not a
mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Cytogenicity in vivo:
A micronucleus assay according to OECD guideline 474 and GLP requirements was performed with the test substanece. This test is performed to detect both chromosome breaking substances (clastogens) and aneuploidy inducing substances (aneugens). These effects are manifested by the formation of micronuclei in polychromatic erythrocytes (PCEs) from bone marrow cells in vivo. The animals were treated once with the highest tolerated dose of the test substance, 5000 mg/kg (as determined in the tolerability test), and the appropriate treatment groups were sacrificed 16, 24 and 48 hours thereafter. Subsequently their femoral bone marrow erythrocytes were scored for micronuclei. The evaluation of the bone marrow smears showed no statistically significant increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at the respective sampling times. It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance.
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. As a result the substance is not considered to be classified as genotoxic under Regulation (EC) No 1272/2008.
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