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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

non mutagenic

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

IN VITRO BACTERIAL REVERSE MUTATION TEST

Two AMES studies were performed using in the first test a plate-incorporation method and in the second one the pre-incubation method.

The first study was performed to investigate the potential of test item to induce gene mutations according to the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100, according to the OECD Guideline 471 (1983) and method B.14 EEC-Directive 92/69 EEC. The assay was performed using a plate incorporation system in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at five concentrations from 10.0 to 5000.0µg/plate.

Toxic effects evidenced by a reduction in the number of induced revertants, occurred in the strains TA 1535 with (exp. I) and without S9 mix (exp.I and II), and in TA 100 without S9 mix (exp. II) at the highest investigated dose.

The plates incubated awith the test article showed normal background growth up to 5000.0µg/plate with and without S9 mix in all strains used.

A significant, concentration-dependent and reproducible increase in the number of revertants was found in the strains TA 1535, TA 1537, TA 98, and TA 100 with and without metabolic activation.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced point mutations by base pair changes and frameshifts in the genome of the strains used.

Therefore, the substance is considered to be positive in this Salmonella typhimurium reverse mutation assay.

The second study was performed to investigate the potential of test item to induce gene mutations according to the pre-incubation test for azo dyes using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100, according to OECD Guideline 471 (1983) and method B.14 EEC-Directive 92/69 EEC. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at five concentrations from 10.0 to 5000.0 µg/plate. Toxic effects, evidenced by a reduction of induced revertant rates occurred at the highest investigated dose in strain TA 1535 with and without metabolic activation in experiment I and II and in strain TA 100 with metabolic activation in experiment I. A significant and reproducible dose-dependent increase in the number of revertants was found in strain TA 1537, TA 98 and TA 100 (exp. II) up to the highest investigated dose, and in strain TA 1535, and TA 100 (exp I) up to 1000 µg/plate all with and without metabolic activation. The plate incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies, In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced point mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100 with and without metabolic activation. Therefore, the substance is considered to be positive in thisSalmonella typhimuriximreverse mutation assay.

IN VITRO MAMMALIAN CELL GENE MUTATION TEST USING THE HPRT GENE

The study was performed to investigate the potential of test item to induce gene mutations at the HGPRT locus in Ovary cells of the Chinese hamster (CHO) in vitro, according to OECD Guideline 476 (1984).

The assay was performed in three and four independent experiments with and without metabolic activation with liver microsomes, respectively, using identical procedures.

In the first experiment the test article was tested with the following concentrations:

without S9 mix: 0.1; 0.3; 0.6; 1.0; 2.5 and 5.0 mg/ml

with S9 mix: 0.1; 0.3; 0.6; 1.0; 2.5 and 5.0mg/ml

In the following experiments the test article was tested with the

following concentrations:

without S9 mix; 0.06; 0.10; 0.30; 0.60; 0.80 and 1.00 mg/ml

with S9 mix: 0.10; 0.30; 0.60; 1.00 and 1.75 mg/ml

According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects. The highest concentrations produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.

Up to the highest investigated dose no relevant increase in mutant colony numbers was obtained in three independent experiments. In the second experiment a high mutation rate of 37.9 mutants/10^6 cells was observed at 1.0 mg/l. This is most probably due to random selection effects at high toxicity (plating efficiency below 1 %, see table IV; poor growth at subcultivation, data not shown). This increase is concluded to be irrelevant and at the other concentrations no increase in mutant colony numbers was observed.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in CHO cells.

Therefore, the substance is considered to be negative in this HGPRT assay.

IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST

The test article was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitro, according to OECD guideline 473 (1983).

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h.

In each experimental group two parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

without S9 mix;                         with S9 mix;

7 h: 0.10 mg/ml                         7 h: 0.30 mg/ml

18 h: 0.01; 0.03; 0.10 mg/ml   18 h: 0.03; 0.10; 0.30 mg/ml

28 h: 0.30 mg/ml                     28 h: 0.30 mg/ml       

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.

Treatment with concentrations higher than 0.25 mg/ml (without and with S9 mix) reduced the plating efficiency of the V79 cells.

Also the mitotic index was reduced after treatment with the highest concentration at fixation intervals 7 h and 28 h (without S9 mix) and 7 h and 18 h (with S9 mix).

There was no relevant increase in cells with structural aberrations after treatment with the test article at each fixation interval either without or with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.

Therefore, the substance is considered to be negative in this chromosomal aberration test.

IN VIVO MICRONUCLEUS TEST

This study was performed to investigate the potential of test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, according to OECD Guideline 474 (1983). The test article was dissolved in water. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 4000 mg/kg b.w.

In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency .

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008 a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms. For the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are: - Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or - Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The test substance did not show any reasons of concern in the tests performed. In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) no. 1272/2008.