Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was negative in guideline compliant Ames and HPRT assays.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No DNA damaging potential was found in a murine micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

A GLP compliant bacterial reverse mutation test was performed according to OECD 471, EPA OPPTS 870.5100 and EU method B.13/14. The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). Two different tests (Ames standard plate test and Prival preincubation test) were performed in two independent experiments in the presence and absence of S9 mix (rat liver S9 mix for standard plate test and hamster liver S9 mix for Prival preincubation test). Parallel with each experiment with and without S-9 mix, a negative control (vehicle control, sterility control) was used for each tester strain in order to determine the spontaneous mutation rate. In both the standard plate test and the Prival incubation test the test substance was checked at the concentrations 33, 100, 333, 1000, 2500 and 5000 µg/plate in absence and presence of S9 mix in the tester strains TA 1535, TA 1537, TA 100, TA 98 and Escherichia coli WP2 uvrA. An increase in the number of revertant colonies by the test substance was not observed in the Ames standard plate test or in the Prival preincubation test, neither without S9 mix nor after the addition of a metabolizing system. Besides, the results of the negative and positive controls, performed in parallel, corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. 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 both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Ames standard plate test and in the Prival preincubation test in the absence and the presence of metabolic activation.

 

HPRT Assay

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to the results of the present in vitro study, the test substance did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and close to the range of the 95% control limit of the historical negative control data, except at two test groups in the 2nd Experiment in the absence of S9 mix which were judged as invalid. The two lowest applied test substance concentrations in the 2nd Experiment without metabolic activation led to unexpectedly high mutant frequencies which were statistically significant increased compared to the respective vehicle control value and clearly exceeded the historical negative control data range. These observations were inconsistent to the data of the 1st Experiment under similar experimental conditions. Additionaly, the reliability of the data is questionable due to the almost inverse dose-relation in this case. In general, a relevant increase in mutant rates occurs at the highest concentrations, often paired with cytotoxic effects. Besides, in a different study performed in the same time period in the laboratory (internal data) a comparable observation means clearly increased values from the lowest to the highest applied test substance concentration including the negative control group was obtained. Thus, it has to be speculated that a technical error occurred by using e.g. improper test culture material for these two test groups. Therefore, the findings at these test groups were considered biologically irrelevant.

When excluding both invalid test groups, in this study in the absence and presence of metabolic activation the values for the corrected mutant frequency were within or close to the range of the concurrent vehicle control values and they were nearby or within the range of the 95% control limit of the historical negative control data. Additionally, no statistically significant increases compared to the respective vehicle control values were determined, and no statistically significant dose-dependent increase in mutant colonies was observed in any experimental part of this study. The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, in the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

 

In vivo Micronucleus

The test item was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in de-ionized water, which was also used as vehicle control. The volume administered orally was 10 ml/kg body weight. 24 h and 48 h after a single administration of the test item 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. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg body weight. 48 h preparation interval: 2000 mg/kg body weight. As estimated by a pre-experiment 2000 mg test item per kg b.w. (the maximum guideline-recommended dose) was suitable. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test article had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was not statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with test material were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008.