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Key value for chemical safety assessment

Additional information

There are reliable in vitro and in vivo studies available to assess the potential of the test substance for gene mutations in bacteria, cytogenicity in mammalian cells and chromosome aberration in mice. For gene mutation in mammalian cells there is only a study with the analogous test substance CAS 36888-99-0 available.

 

Gene mutation in bacteria:

In the Ames test comparable to OECD guideline 471, the test 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 1538, TA 98 (BASF SE, 1980). The study was performed prior to the introduction of GLP and the OECD testing guideline, but is comparable both in design and reporting. In deviation to the current version of the OECD testing guideline 471, the design did not include a tester strain for crosslinking. The investigations were performed with Aroclor 1254-induced rat liver S-9 mix and without microsomal activation at a dose range up to 2500 µg/plate. At the highest dose of 2500 µg/plate (the limit of complete solubility).

A weak bacteriotoxic effect (slight decrease in the number of his+revertants) was observed with some strains at 2500µg/plate.

An increase in the number of his+revertants could not be observed either without S-9 mix or after addition of a metabolizing system. Therefore, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

 

In a GLP conform study conducted according to OECD guideline 471, the potential of the test substance (purity: 99.3 weight-%) to induce gene mutations was investigated in Salmonella typhimurium strains TA 100, TA 1535, TA1537 and TA 98, and in Escherichia coli WP2uvrA (Mitsubishi 1999). The test was performed with (phenobarbital and benzoflavone induced rat liver S9 -mix) and without microsomal activation at a dose range up to 5000 µg/plate. At 19.5 µg/plate and over in the absence of S9 mix and at 78.1 µg/plate in the presence of S9 mix, precipitates were observed on the agar plates. Microbial toxicity was not observed in any of the tester strains regardless of the presence or absence of S9 mix.

The number of revertant colonies was smaller than twice that of the corresponding negative (solvent) control in all the tester strains regardless of the presence or absence of S9 mix. These results suggest that the test substance is not mutagenic under the conditions of the study.

Gene mutation in mammalian cells:

In a GLP conform study conducted according to the OECD guideline 476 the potential of the analogous test item CAS 36888-99-0 (purity: > 99 weight-%) to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro was assessed (BASF SE, 2012). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, doses in the range from 0 – 400 µg/ml were tested in this study.

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. 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, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

The analogous test substance was poorly soluble in the most suitable vehicle culture medium. Due to lacking distinct cytotoxicity in the pretests in the absence and presence of metabolic activation after 4 hours exposure all experimental parts with short-term exposure were performed using concentrations at the border of saturation of culture medium. In the pretest with 24 hours exposure in the absence of metabolic activation cytotoxicity of about 20% relative survival was observed at intermediate concentrations showing strong test substance precipitation.

The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the analogous 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 mammalian cells:

 

In a GLP conform study conducted according to OECD guideline 473, the test substance (purity: 99.3 weight-%) was assessed in vitro for possible clastogenic activity both in the presence and in the absence of a metabolizing system (S9 mix) using the established cell line (CHL/IU) from the lungs of a female Chinese hamster (Mitsubishi 2000).

In a preliminary test, concentrations of 313, 625, 1250, 2500, and 5000 µg/mL (pulse treatment) or 156, 313, 625, 1250, 2500, and 5000 µg/mL (continuous treatment), in the presence or absence of S9 mix, were used to evaluate the cell growth inhibition. No inhibit of cell growth higher than 50 % was observed, both in the pulse and in the continuous treatment protocols.

Therefore, the concentration levels selected for the main chromosomal aberration test were 1250, 2500, and 5000 µg/mL with and without S9 mix. The incidence of cells with structural and numerical aberrant chromosomes was less than 5% in each treatment. The test substance was judged as negative in the pulse treatment and a second test with continuous treatment.

It was concluded that the test item did not have a clastogenic potential in this CHL/IU cell line system.

 

Cytogenicity in vivo

 

For the determination of cytogenicity in vivo the substance CAS 36888-99-0 is used for read-across.

A micronucleus assay conducted according to OECD guideline 474 and GLP requirements was performed with the test item CAS 36888-99-0 (purity: > 99%) (BASF SE, 2008). This test is performed to detect both chromosome breaking substances (clastogens) and aneuploidy inducing substances (aneugens) in NMRI mice.

For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 500, 1000 and 2000 mg/kg body weight in a volume of 20 mL/kg body weight. A control group received the vehicle by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. Only the 24 hour sacrifice interval was investigated in the test groups of 1000 and 500 mg/kg body weight and in the positive control groups. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

The rate of micronuclei was always close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the analogous test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Short description of key information:
in vitro:
Gene mutation in bacteria:
key study, Ames test, S. typhimurium, with and without metabolic activation: negative (non-GLP, comparable to OECD 471; BASF SE, 1980)
key study, Ames test, S. typhimurium, with and without metabolic activation: negative (GLP, OECD 471; Mitsubishi 1999)

Gene mutation in mammalian cells:
read-across, CAS 36888-99-0, key study, HPRT test, CHO cells, with and without metabolic activation: negative (GLP, OECD 476; BASF SE, 2012)

Cytogenicity in mammalian cells:
key study, Chromosome aberration, chinese hamster lung cell line CHL/U: negative (GLP, comparable to OECD 473; Mitsubishi 2000b)

in vivo:
read-across, CAS 36888-99-0, key study, micronucleus test, mouse, in vivo: negative (GLP, OECD 474; BASF SE, 2008)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).