Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Read across justification

The potential to cause gene mutations in bacterial and mammalian cells and formation of micronuclei in vivo was assessed on a structural analogue in GLP-compliant tests according to OECD guidelines 471, 476 and 474. Both substances are calcium salts and share high similarity in structure; they differ in the presence of one chlorine atom only. Moreover, the analogue has a higher molecular weight and both substances are insoluble in water. Therefore, it is acceptable to derive information about genotoxicity from the data of the analogue substance. A detailed read across justification is given in Annex I of the CSR.

Performance and observations

An Ames test was performed to investigate the potential of the test item to induce gene mutations using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100 (Ciba, 1979). The assay was performed without preval modification and in presence and absence of S9-mix from Aroclor induced rat liver. The test item, dissolved in DMSO, was tested at five concentrations in the range of 25 and 2025 µg/plate. No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

Read across to CAS 71832 -85 -4

This study (Ciba, 1997) was performed to investigate the potential of the test item to induce gene mutations using the S. typhimurium strains TA1535, TA 1537, TA 1538, TA 98, and TA 100, and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and with and without preval modification. Each concentration, including the controls, was tested in triplicate. The test item was tested at five concentrations in the range of 2.4 and 1500 µg/plate (experiment I) and 93.75 to 1500 µg/plate (experiment II).

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) or by prival modification.

Read across to CSA 71832 -85 -4

In the second study (Ciba, 1997b), mouse lymphoma cells (L5178Ytk+/-) were treated with 11.25 to 180 µg/ml of the test item in presence or absence of S9-mix from Aroclor induced rat liver. The assay was performed in two independent experiments; each concentration was tested in triplicate. Two days after treatment, all doses tested, were selected to determine viability and 5-trifluorothymidine resistance. Precipitation was observed at the top two dose levels in each experiment, toxicity did not occur. No significant increases in mutant frequency were observed following treatment with the test item at any dose level in the absence or presence of S-9, in both experiments.

 Read across to CAS 71832 -85 -4

The test article was assayed in vivo in a mouse bone marrow micronucleus test at three dose levels. The choice of dose levels was based on an initial toxicity range-finding study. The test substance was administered intraperitoneally at 100, 200 and 400 mg/kg/day to groups of five male and five female mice killed 24 or 48 hours after the second administration. Cyclophosphamide (CPA) served as positive control and was administered as a single dose at 40 mg/kg to groups of five male and five female mice which were killed after 24 hours. Both femurs of each animal which was exposed were removed and bone marrow extracted to prepare slides with air dried bone marrow smear. Slides from all dose groups were analysed.

Clinical signs of toxicity were present in all treated animals. Eye closure was apparent at 100 and 200 mg/kg/day. Prostration, hunched appearance, piloerection, coldness, eye closure and twisting were seen among animals dosed at 400 mg/kg/day. Three males and two females receiving this dose were either found dead or were killed in extremis prior to sampling. Loss of body weight was seen at all dose levels.

Microscopic analysis of the slides revealed normal group mean ratios of PCE (polychromatic erythrocytes) to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE at the control group. Mice treated with the test articel exhibited mean ratios of PCE to NCE which were generally lower than those in concurrent controls indicating exposure of the target tissue to the test article. Frequencies of micronucleated PCE were similar to control groups at both sampling times.

 

Discussion

In conclusion, the test item did not induce gene mutations in bacterial cells with and without metabolic activation. Due to the lack of a prival modification of the Ames test, this assay is not sufficient so assess the mutagenic potential of metabolites of an azo reductase reaction. Therefore, additional genotoxicity data were derived from a structural analogue to assess the genotoxic potential. The analogue was negative for gene mutations in an Ames test with preval modification and in a mouse lymphoma assay. The structural analogue did also not induce formation of micronuclei in bone marrow derived erythrocytes from mouse.

Thus, it can be concluded that the test article is not genotoxic.


Short description of key information:
Test on genotoxicity of the test substance were not performed; the accordingly information is derived from experimental data of a structural analogue. An Ames tests, a mutagenicity assay in mammalian cells and a micronucleus assay in vivo were performed (OECD guideline 471, 476 and 474) to evaluate the genotoxic potential of the test substance. The test item or an analogue substance, respectively, did not induce mutations in bacterial or mammalian cells or formation of micronuclei in bone marrow derived erythrocytes from mice. Therefore, the substance is considered as non-mutagenic under the conditions of these tests.

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 genotoxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

 

 

Classification, Labeling, 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 genotoxicity under Regulation (EC) No. 1272/2008, as amended for the second time in Directive (EC 286/2011).