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Additional information

Different studies were available performed whether in vitro or in vivo.

In vitro, three GLP key studies were available.

 

First, in an Ames test performed accordingly the Guideline No. 471, strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to the substance (> 98%) with or without metabolic activation in two independent experiments. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered as valid.

In the absence of S9 mix, a moderate to strong toxicity was noted at dose-levels lower than 5000 µg/plate in all 5 strains. In the presence of S9 mix, in the first experiment, using the direct plate incorporation method, and in the second experiment, using the preincubation method, a moderate to strong toxicity was noted at dose-levels lower than 5000 µg/plate. Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium as no biologically significant increase in the number of revertant nor dose-related effect were observed.

 

Second, in a mammalian cell gene mutation assay (HPRT) performed according to the Guideline No. 476, Chinese hamster V79 cells were exposed to the substance (99% pure) diluted in DMSO both in the presence and absence of mammalian metabolic activation in two independent experiments. The first main experiment was performed with and without metabolic activation and a treatment period of 4 hours. In the second experiment the cells were exposed to the test item for 4 hours with and 24 hours without metabolic activation. The number of mutant for the vehicle and positive controls was as specified in the acceptance criteria. Relevant cytotoxic effects as indicated by a relative cloning efficiency (survival) of less than 50 % in both parallel cultures were observed. No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. Under the experimental conditions, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the substance is not considered to be mutagenic in this HPRT assay.

 

Third, in vitro mammalian chromosomal aberration test, V79 cell cultures of the Chinese hamster were exposed to the substance (99 % pure) in DMSO bothin the presence and absence of metabolic activation (S9-Mix from phenobarbital/ß-naphthoflavone induced rat liver S9).This study was performed according to the OECD Guideline No. 473 and in compliance with GLP.

Test item concentrations between 11.7 and 3000 µg/mL (approx. 10 mM) were chosen for the evaluation of cytotoxicity. Due to strong toxic effects, the experimental part without S9 mix was repeated with test item concentrations between 0.1 and 30.0 µg/mL. Due to missing cytotoxicity this part was repeated once again with test item concentrations between 0.6 and 300.0 µg/mL. No precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated main experiment. Using reduced cell numbers and mitotic indices as an indicator for toxicity in the main experiment, clear toxic effects were observed after treatment with 75.0 µg/mL and above in the absence of S9 mix and with 375 µg/mL and above in the presence of S9 mix.

According to the OECD Guideline only one experiment was performed, since the test item was considered to be mutagenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation.

In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in theabsence of S9 mix, where only 50 metaphases were evaluated.

Neither precipitation of the test item in the culture medium nor relevant increase in theosmolarity or pH value was observed.

Cytotoxicity was measured as reduced cell numbers and mitotic indices. In the absence of S9 mix cell numbers were reduced to 58 % and mitotic index to 41.1 % of control (75 µg/mL). In the presence of metabolic activation cell numbers were reduced to 40.6 % of control and the mitotic index to 68.9 % of control (375 µg/mL). In fact, the total number of mitotic cells was only 23.8% (41.1% mitotic indice of 58% of cell number/control) at the highest concentration (75 g/mL) in the absence of S9 and 28% (at 375 µg/mL) in the presence of S9. Therefore, the highest concentrations showed excessive test item cytotoxicity.

In the absence and presence of S9 mix statistically significant and biologically relevant increases in chromosomal aberrations (4.0, 11.0 and 10.5 %, respectively) were observed after treatment with 37.5 and 75 µg/mL in the absence of S9 mix and with 375 µg/mL in the presence of S9 mix. All values were above the range of the solvent control values (0.5 - 1.0 % aberrant cells, excluding gaps) and clearly exceeded the laboratory’s historical solvent control data (0.0 - 4.0 % aberrant cells excluding gaps). In addition, the number of aberrant cells at these concentrations increased in a dose-related manner. Therefore, this observation has to be regarded as biologically relevant by the authors.

No biologically relevant but statistically significant increase in the rate of polyploid metaphases was found after treatment with the test item (2.0 – 4.7 %) as compared to the rates of the solvent controls (2.9 – 3.3 %).

In the absence and presence of S9 mix the frequencies of polyploid metaphases and endoreduplications were partly statistically significantly increased but were within or close to the range of the laboratory’s historical control data (1.0 – 4.6 % without S9 mix; 0.6 - 4.3 %with S9 mix).Moreover, the frequencies of polyploid metaphases and endoreduplications decreased while the cytotoxicity increased.

Considering the excessive cytotoxicity (see above) at the highest doses with and without S9, the study results may become equivocal in the absence of S9 and negative in the presence of S9.

In both experiments, either EMS (1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test substance induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to strong cytotoxic concentrations. Taking into account the raw data, the substance inhibited mitotic processes and induced numerical chromosome aberrations (polyploidy and endoreduplications) in theV79 cells with and without metabolic activation. However, considering the cytotoxicity at the highest doses which may be considered as excessive with and without S9, the study results may become equivocal without S9 and negative with S9. However, they have to be confirm in an in vivo study.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473 for cytogenicity.

In vivo genotoxicity study has been performed as positive results for clastogenicity were obtained in an in vitro study. Hence, two GLP micronucleus assays (key studies) were available after a single or repeated doses.

Both tests were performed according to the Guideline No. 474 and gave negative results concerning the induction of micronuclei after orally exposure to the substance.

 

In the first micronucleus assay, male NMRI mice were exposed to the substance (98 % pure) diluted in corn oil by a single gavage dose. Different dose levels (75, 150, and 300 mg/kg bw) and two chromosome preparation intervals (24 and 48h) were tested. The mean number of polychromatic erythrocytes (PCEs) was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the substance did not have any cytotoxic properties in the bone marrow.No statistically significant or biologically relevant enhancement was observed in the frequency of the detected micronuclei at any preparation interval and dose level in comparison to the vehicle controls. The positive control showed a statistically significant increase of induced micronucleus frequency; the study was therefore considered as valid.

 

In the second micronucleus assay, male and female Han rats were exposed to the substance (99 % pure) diluted in corn oil by repeated gavage. This second micronucleus assay was part of a combined repeated dose toxicity study with the reproduction / developmental toxicity screening test. Male rats were exposed once daily for 4 weeks and female rats were exposed at least for 7 weeks. Both sexes were exposed to 0; 30; 60 and 90 mg/kg bw/day substance. 24 hours after the final treatment, blood samples were obtained from all males and all females from each group and analysed for micronuclei induction. No relevant effect on the Polychromatic erythrocyte (PCE)/Normochromatic erythrocyte (NCE) ratio was observed after treatment with the test item when compared to the vehicle control. Concerning the micronuclei, there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any dose level of the test item in comparison to the vehicle control. The positive control showed a statistically significant increase of induced micronucleus frequency; the study was therefore considered as valid.

In conclusion, the substance is considered neither mutagenic nor clastogenic.


Short description of key information:
In vitro: Ames test (negative), Mouse lymphoma assay (negative), Chromosome aberration test (positive)
In vivo: Micronucleus test after a single exposure (negative), micronucleus test after repeated dose exposure (negative)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No harmonised classification is available.

Based on the results of the in vivo key studies, no indications of genotoxicity were observed. Therefore according tothe criteria of the Regulation (EC) No. 1272/2008 (CLP) and the Council Directive 67/548/EEC (and subsequent adaptations) no additional self-classification is proposed for the substance.