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Additional information

Gene mutation in bacteria:

In an available Ames test conducted comparable to OECD guideline 471, the 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 and Escherichia coli WP2 uvrA (BASF SE, 1982). The investigations were performed with (Aroclor 1254-induced rat liver S-9 mix) and without microsomal activation at a dose range up to 5000 µg/plate. From 500 µg/plate onward precipitates of the test substance were visible in the agar.

In the experiments performed without and with microsomal activation, none of the tested concentrations of the test item led to an increase in the incidence of both histidine- or tryptophan-prototrophic mutants in comparison with the negative control. No evidence of the induction of point mutations by the test item or by the metabolites of the substance was found.

According to the results of the present study, the test substance is thus not mutagenic in the Ames test under the experimental conditions chosen.

 

In a GLP conform study conducted according to OECD guideline 471, the potential of the test substance to induce gene mutations was investigated using the Salmonella typhimurium strains TA 100, TA 1535, TA1537 and TA 98 (Clariant, 1996).

Two independent mutagenicity studies were conducted, each in the absence and in the presence of a metabolizing system derived from rat Iiver homogenate. For both studies the compound was suspended in DMSO, and each bacterial strain was exposed to 6 dose levels. Doses for both studies ranged from 4 to 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All positive control compounds gave the expected increase in the number of revertant colonies.

No toxicity was found in the mutagenicity experiments with and without metabolic activation.

In the toxicity test using histidine enriched agar plates and a dilution of the tester strain TA 100 (designated TA 100 D), which was performed in parallel with the second experiment, the test compound proved to be not toxic to the bacterial strain.

In the absence and in the presence of the metabolic activation system did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that the test substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

 

Gene mutation in mammalian cells:

In a GLP conform study conducted according to the OECD guideline 476 the potential of the test item (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 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 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:

For the determination of chromosome aberration in mammalian cells the substance CAS 76199-85-4 is used for read-across.

In a GLP conform study conducted similar to OECD guideline 473, the analogous test substance CAS 76199-85-4 (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/U) 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 analogous test substance was judged as negative in the pulse treatment and a second test with continuous treatment.

It was concluded that the test item CAS 76199-85-4 did not have a clastogenic potential in this CHL/IU cell line system.

 

Cytogenicity in vivo

A micronucleus assay conducted according to OECD guideline 474 and GLP requirements was performed with the test item (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 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:
There are no data gaps in regard to mutagenicity. Pigment Yellow 139 is not mutagenic in bacteria and mammalian cells in vitro and not genotoxic in the micronucleus test in vivo. The semi condensate Pigment Yellow 185 is not mutagenic in bacteria and not clastogenic in mammalian cells in vitro.

in vitro:
Gene mutation in bacteria:
key study, Ames test, S. typhimurium/ E.coli, with and without metabolic activation: negative (non-GLP, comparable to OECD 471; BASF SE, 1982)
supporting study, Ames test, S. typhimurium, with and without metabolic activation: negative (GLP, OECD 471; Clariant, 1996)

Gene mutation in mammalian cells:
key study, HPRT test, CHO cells, with and without metabolic activation: negative (GLP, OECD 476; BASF SE, 2012)

Cytogenicity in mammalian cells:
read-across CAS 76199-85-4, key study, Chromosome aberration, chinese hamster lung cell line CHL/U, in vitro: negative (GLP, comparable to OECD 473; BASF SE, 2000)

in vivo:
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).

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