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In vitro data

Reverse mutation in bacteria:

The potential of the test material to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines OECD 471, EU Method B.14 and EPA OPPTS 798.5265. Five strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) and one Escherichia coli strain (WP2uvrA) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). The first confirmatory experiment was carried out with and without metabolic activation. Since marked differences in toxicity occurred with the strains used, a second confirmatory experiment was performed with and without metabolic activation on strains TA 1535 and TA 102. Strain TA 98 was tested without activation. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

The original experiment with and without metabolic activation and the confirmatory experiment without activation were performed as standard plate incorporation assay. The first and second confirmatory experiments with metabolic activation were carried out as preincubation assay.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of test material led to an increase in the incidence of either histidine- or tryptophan-prototrophic mutants by comparison with the solvent control.

 

Gene mutation in mammalian cells:

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476and EPA OPPTS 798.5300. V79 Chinese hamster cells were treatedin vitroboth in the presence and absence of a rat liver derived metabolic system (S9 mix).

A preliminary range finding test was run assessing cytotoxicity. The test material was tested up to a maximum concentration of 4000 µg/mL, representing the solubility limit of the test material. In the part with metabolic activation the test material was found to be practically not toxic. Without metabolic activation treatment with test material led to a moderate and concentration-dependent growth inhibiting down to the concentration of 500 µg/mL. The next lower concentration of 250 µg/mL was not toxic. Accordingly, 4000 µg/mL with and without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

 The original mutagenicity assay was therefore performed at 148.15, 444.44, 1333.33 and 4000.00 µg/mL. The test material exerted practically no toxicity after treatment and expression in the test with metabolic activation. In the confirmatory experiment with metabolic activation the concentrations applied were 500.00, 1000.00, 2000.00 and 4000.00 µg/mL. Again, the test material proved not toxic after treatment and expression. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test material revealed no biologically relevant increase of the mutant frequencies as determined by screening with 6-thioguanine.

In the original experiment without metabolic activation, the mean growth inhibition value found at the highest concentration after treatment was 59.6%. After the expression period, this concentration was practically not toxic. In the confirmatory experiment without metabolic activation the highest concentration revealed a mean acute growth inhibitory effect of 57.7%. No toxicity was observed after the expression period.

 In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test material revealed no biologically relevant increase of the mutant frequencies as determined by the screening with 6-thioguanine.

 Based on the results of two independently performed experiments and under the given experimental conditions, it was concluded that the test material did not show any mutagenic activity in the forward mutation assay.

In vitro chromosome aberration:

The potential of the test material to induce structural chromosomal aberrations was determined in a study performed in accordance with standardised guidelines OECD 473, EU Method B.10 and EPA OPPTS 798.5375. In each experimental group two parallel cultures were analysed, in the absence and presence of S9 mix, with 100 metaphases per replicate culture being scored for structural chromosomal aberrations. The highest applied concentration in this study was 4000 μg/mL; final concentrations higher than 4000 µg/mL of culture medium could not be achieved due to solubility limitations. Concentration selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of test material precipitation in accordance with the guidelines followed. In the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test material. Under the specific conditions of this assay, the test material did not induce structural chromosomal aberrations as determined by the chromosome aberration test in Chinese Hamster Ovary cells in vitro. Therefore, the test material is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation.

 

Gene mutation in mammalian cells:

The potential of the test material to cause gene mutation or clastogenic effects in mammalian cells was determined in accordance with standardised guidelines OECD 476and EPA OPPTS 798.5300. V79 Chinese hamster cells were treated in vitro both in the presence and absence of a rat liver derived metabolic system (S9 mix).

A preliminary range finding test was run assessing cytotoxicity. The test material was tested up to a maximum concentration of 4000 µg/mL, representing the solubility limit of the test material. In the part with metabolic activation the test material was found to be practically not toxic. Without metabolic activation treatment with test material led to a moderate and concentration-dependent growth inhibiting down to the concentration of 500 µg/mL. The next lower concentration of 250 µg/mL was not toxic. Accordingly, 4000 µg/mL with and without metabolic activation were chosen as highest concentrations for the first mutagenicity assay.

 The original mutagenicity assay was therefore performed at 148.15, 444.44, 1333.33 and 4000.00 µg/mL. The test material exerted practically no toxicity after treatment and expression in the test with metabolic activation. In the confirmatory experiment with metabolic activation the concentrations applied were 500.00, 1000.00, 2000.00 and 4000.00 µg/mL. Again, the test material proved not toxic after treatment and expression. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test material revealed no biologically relevant increase of the mutant frequencies as determined by screening with 6-thioguanine.

In the original experiment without metabolic activation, the mean growth inhibition value found at the highest concentration after treatment was 59.6%. After the expression period, this concentration was practically not toxic. In the confirmatory experiment without metabolic activation the highest concentration revealed a mean acute growth inhibitory effect of 57.7%. No toxicity was observed after the expression period.

 In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test material revealed no biologically relevant increase of the mutant frequencies as determined by the screening with 6-thioguanine.

 Based on the results of two independently performed experiments and under the given experimental conditions, it was concluded that the test material did not show any mutagenic activity in the forward mutation assay.

 

DNA damage and/or repair:

The mutagenic potential of the test material to cause DNA repair was assessed in accordance with standardised guidelines OECD 4832, EU Method B.18 and EPA OPPTS 798.5550. The test material was dissolved in DMSO and tested at concentrations from 7.82 - 250.0 µg/mL (original experiment) and at concentrations from 0.25 - 250.0 µg/mL (confirmatory experiment). Under the conditions of the study, the test material exerted a concentration-dependent toxicity at the upper concentrations. The highest concentration of 250.0 µg/mL reduced cellular viability by about 70%. In both experiments performed the test material did not induce a significant increase in the DNA repair activity of primary rat hepatocytes. Hence, no evidence of induction of DNA damage by the test material was obtained that could be interpreted as suggestive of a genotoxic property of the test material in vitro.

 

In vivo data

In vivo chromosome aberration:

The potential of the test material to cause clastogenic and/or aneugenic effects on mouse bone marrow cells in vivo was determined in accordance with standardised guidelines OECD 474, EU Method B.12 and EPA OPPTS 798.5395. The test material was administered once by gavage to groups of 5 male and 5 female mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone or with a positive control. From the high dose group and from the vehicle control group animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose groups and from the positive control group animals were sacrificed at 24 hours after administration. Subsequently, femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

The high dose applied caused significant toxicity at sampling times of 16 and 24 hours after treatment. Ataxia was observed with all animals at the time of sacrifice. At 48 hours sampling time ataxia was observed with two females.

In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective vehicle control group. Under the conditions of the study no evidence for clastogenic or aneugenic effects were obtained in mice treated with the test material.

 

All studies were performed in line with GLP and in accordance with standardised guidelines with a high standard of reporting. The studies were assigned a reliability score of 1 in accordance with the criteria for assessing data quality as outlined in Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

 

The available data are considered to be complete and the conclusion, non-mutagenic, was taken forward for risk assessment.


Justification for selection of genetic toxicity endpoint
No study was selected as the key study for this endpoint, as there were several high quality studies available which investigated different aspects of genetic toxicity and are therefore not comparable.

Short description of key information:
IN VITRO DATA
Reverse mutation in bacteria: Negative (S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 and E. coli WP2uvrA in the presence and absence of S9-mix) OECD 471, EU Method B.14, EPA OPPTS 798.5265 - Deparade 1997

In vitro chromosome aberration: Negative, Chinese hamster ovary cells (CHO) with and without metabolic activation, OECD 473, EU Method B.10, EPA OPPTS 798.5375 - Ogorek 1998a

Gene mutation in mammalian cells: Negative, V79 Chinese hamster cells with and without metabolic activation, OECD 476, EPA OPPTS 798.5300 - Ogorek 1997

DNA damage and/or repair: Negative, rat hepatocytes, OECD 482, EU Method B.10, EPA OPPTS 798.5550 - Ogorek 1998b

IN VIVO DATA
In vivo chromosome aberration: Negative, femur bone marrow, OECD 474, EU Method B.12, EPA OPPTS 798.5395 - Deparade 1997

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Point 3.5 of Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the negative response noted in all the available genetic toxicity studies.

In accordance with criteria for classification as defined by Directive 2001/59/EC, Annex VI, Point 4.2.2, the test material does not require classification as mutagenic based on the negative response noted in all the available genetic toxicity studies.

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