Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test substance is mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT: 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.

in vitro Micronucelus Assay: The test item is considered to be non-mutagenic in the in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The substance was tested according OECD TG 471 for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The following strains were used: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA.

A Standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats) was performed using a dose range from 33 μg up to 6250 μg/plate.

Precipitation of the test substance was found from about 1000 μg/plate onward with and without S9 mix. A weak bacteriotoxic effect was observed only using the tester strain E. coli with S9 mix at a concentration of 6250 μg/plate.

A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test either with or without S9 mix using tester strains TA 1535 and E.coli WP2 uvrA. A distinct and partly dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation. In the strain TA 100 without S9 mix an increase of revertants at concentrations of 333, 1000, 3125 and 6250 μg/plate and with S9 mix an increase of revertants at concentrations of 100, 333, 1000, 3125 and 6250 μg/plate was detected. In TA 98 with and without S9 mix an increase of revertants in all tested concentrations was observed. Using tester strain TA 1537 a relevant and partly dose depending increase in the number of his+ revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed with and without metabolic activation. In TA 1537 with and without S9 mix an increase of revertants at concentrations of 333, 1000, 3125 and 6250 μg/plate was detectable with S9 mix.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies within or above the range of the historical positive control data.

Under the experimental conditions of this study, the test substance is strongly mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (BASF, 2017).

HPRT

The test item was assessed according OECD TG 476 for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats.

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, the following concentrations were tested:

1st Experiment

without S9 mix 0; 7.81; 15.63; 31.25; 62.50; 125; 250; 500 μg/mL

with S9 mix 0; 3.91; 7.81; 15.63; 31.25; 62.50; 125; 250 μg/mL

2nd Experiment

with S9 mix 0; 3.91;7.81; 15.63; 31.25; 62.50; 125; 250 μg/mL

3rd Experiment

with S9 mix 0; 3.91; 7.81;15.63; 31.25; 62.50; 125; 250 μg/mL

4th Experiment

without S9 mix 0; 6.25; 12.50; 25;50; 62.50; 75; 150 μg/mL

with S9 mix 0; 12.50;25; 50; 75; 100;125; 150; 300 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, 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, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In the absence of metabolic activation, cytotoxicity was observed at the highest concentrations evaluated for gene mutations. In the presence of metabolic activation, no significant cytotoxicity could be observed up to precipitation levels.

Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.

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 (BASF, 2018).

In vitro Micronucleus test

The test item suspended (Exp. I) or dissolved (Exp. IIA and IIB) in DMSO, was assessed according OECD TG 487 for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following concentrations were tested:

Experiment I: 18, 31.4, 55, 96.3, 169, 295, 616, 903, 2710 µg/mL

Experiment IIA: 150 µg/mL

Experiment IIB: 150 µg/mL

The Exposure period was 4 hours in Experiment I (with and without S9 mix), 20 hours in Experiment IIA+B (without S9 mix) and 4 hours in Experiment IIA with S9 mix.

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2710 μg/mL of the test item) was chosen with regard to the purity (73.8%) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In Experiment I in the absence of S9 mix, clear cytotoxicity was observed in the highest evaluated concentration. In Experiment IIB in the absence of S9 mix, again clear cytotoxicity was observed in the highest evaluated concentration. The cytotoxicity observed was not reflected in the cytostasis (39.2 % after treatment with 55.6 μg/mL) as the CBPI pictures cytotoxicity by measuring proliferation and may not detect cytotoxic events like necrosis and apoptosis. The cytotoxicity was judged in the course of a microscopical pre-check for guideline requested quality and quantity criteria. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations (Envigo, 2017).

General conclusion

Here, the substance was mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay (Ames test) in the absence and the presence of metabolic activation. On the contrary, 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 and non-mutagenic in the in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Kirkland et al. (Mutation Research 775-776 (2014) 69-80) published an article in which the authors conclude that: “Thus, in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential."

Therefore, the test item is not considered to induce genotoxicity and the presented studies and data are sufficient evidence to waive the need for in vivo gene mutation testing.

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.