Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Ames-Test:

Key study: In order to assess its mutagenic potential, the registered substance was evaluated in a GLP-study according to OECD test guideline 471 (Ames). The present study was conducted by two methods, Direct plate incorporation method and pre-incubation method with five tester strains of Salmonella typhimurium (TA 98, TA 102, TA 100, TA 1535 and TA 1537).The registered substance dissolved in DMSO was tested at the doses of 5.0, 2.5, 1.25, 0.62 and 0.311 µL/plate. Simultaneously, cultures in control either received DMSO or positive mutagens (Benzo (a) pyrene, 2-Nitrofluorene, Sodium azide, 9-Aminoacridine and Mitomycin C). In order to study the role of metabolic activation, cultures were incubated with or without S9 mix. The induction of Histidine positive colonies was computed and results were statistically treated for comparison. The study indicated lack of statistically significant induction of His' revertant colonies in any of the tester strains either with or without S9 addition in the culture. Based on the above results, it is concluded that registered substance is non-mutagenic by Ames bacterial reverse mutation assay (IIBAT, 2006).

In a supporting GLP-study according to OECD test guideline 471 (Ames: only strain TA102), the registered substance did not induce a mutagenic response and is therefore considered negative (IIBAT, 2007).

Chromosome Aberration:

The registered substance, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to OECD test guideline 473 under GLP-conditions. The following study design was performed:

 

Without S9 mix

With S9 mix

Exp. I

Exp. II

Exp. I

Exp. II

Exposure period

4 h

18 h

28 h

4h

2 h

Recovery

14 h

-

-

14 h

24 h

Preparation interval

18 h

18 h

28 h

18 h

28 h

In each experimental group two parallel cultures were set up. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in Experiment II, without metabolic activation, at preparation interval 28 hrs, where only 50 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (2840 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In this study, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentrations being far in the range of test item precipitation.

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. Although dose-related increases in the number of aberrant cells were observed in Experiments I and II, in the absence of S9 mix and the number of aberrant cells was significantly increased at two concentrations in Experiment II, in the absence of S9 mix, these observations have to be regarded as biologically irrelevant, since all values were within the range of the historical control data (0.0 – 4.0 % aberrant cells, exclusive gaps).

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

As the registered substance did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro, it is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix when tested up to precipitating concentrations (RCC, 2006)

Mammalian cell gene mutation:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration in the pre-experiment (2840 μg/mL) was equal to a molar concentration of about 10 mM. The test item was dissolved in ethanol.

The tested concentrations of the main experiments were 11.1, 22.2, 44.4, 88.8, 177.5, 355.0 µg/mL. The concentration range of the main experiments was limited by the occurrence of phase separation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in any of the main experiments. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in this HPRT assay. (Harlan, 2012)


Short description of key information:
In a GLP-study according to OECD test guideline 471 (Ames), the registered substance did not induce a mutagenic response and is therefore considered negative.
In a GLP-Study according to OECD test guideline 473, the registered substance did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro and is therefore considered negative.
In a GLP-study according to OECD test guideline 476, the registered substance dic not induce gene mutations at the HPRT locus in V79 cells (Chinese Hamster cell line) in vitro and is therefore considered negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative in vitro results of the Ames test, the Chromosome aberration assay, and the mammalian cell gene mutation test, the test item has not to be classified and labelled mutagenic according to Regulation (EC) No. 1272/2008 (GHS/CLP) and EU Directive 67/548/EEC (DSD).