Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Read across justification

There are only limited data available on genotoxicity of the test item. Therefore, it is acceptable to derive the information on mutagenicity and clastogenicity from experimental data of a structural analogue (di-ammoinium salt) since both are salts with similar structures and comparable solubility (a detailed read across justification is given in CSR, Annex I).

Performance and observations

There are two reliable, GLP-conform in vitro studies available to assess the potential of the substance for gene mutations in bacteria and clastogenicity in mammalian cells.

The first study (BASF AG 2002b) was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100, TA 1538 and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without S9 mix from aroclor induced rat liver and non-induced Syrian hamster liver. The test item, dissolved in DMSO, was tested at concentrations of 22 - 5500 µg/plate (standard test) and 4 - 2500 µg/plate (prival modification test). 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. Precipitation occured from 500 µg/plate onward.

In a second study (BASF AG 2002c), the test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells in two independent experiments. The experiments were carried out at 0 - 500 µg/plate, 4h exposure and with or without metabolic activation. The test article caused a significant, biological relevant increase in structural chromosomal aberrations in the presence of S-9 mix. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells and demonstrated the sensitivity of the test compound. Thus, the test substance is considered to be clastogenic under the in vitro conditions of this assay using V79 cells.

In addition to the chromosome aberration asssay in vitro, a 1 : 1 mix of the calcium salt and a corresponding sodium salt was tested in vivo for induction of micronuclei in mouse (BASF AG 2005). Based upon the results of the acute oral toxicity study, two doses of 500, 1000 and 2000 mg/kg bw were administered by oral gavage in a 24 h intervall to five male animals. Animals were sacrificed and samples of bone marrow were taken 24h after the second administration. Bone marrow smears were made, air dried and dyed with Giemsa solution. 2000 polychromatic erythrocytes per mouse were scored for incidence of micronuclei. Positive control experiments were performed with cyclophosphamide and vincristine. The bone marrow smears from treated animals showed no increase in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals. Inhibition of eyrthropoisis was also not detected.

At last, the substance (analogue diammonium salt) 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 (BASF 2011). Three experiments were carried out independently of each other at dose levels of 2.5 - 5200 ug/ml in absence and presence of a metabolic activation syten for 4h and 24h, respectively. Thereafter, 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. Precipitation occurred from the lowest applied concentration onward. In all experimental parts in the absence of S9 mix cytotoxicity was observed at strongly precipitating concentrations only. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system. In the 1st Experiment after 4 hours exposure in the absence of S9 mix an increase in the number of mutant colonies was observed in a single culture which was not corroborated in the culture treated in parallel or in the repeat experiment (2nd Experiment).

Discussion

The test item as well as analogue substances were tested for genotoxicity in vitro and in vivo. The test item was negative in a modified Ames test and positive for chromosome aberrations in V79 cells. However, a micronulcei test in mice yielded a negative result. Structural chromosome aberrations in V79 cells may be explained by precipitation of the test item from 125 microgramm onward leading to cytotoxicity induced DNA-damages. Moreover, a description on analytical purity and impurities was not given. Thus, it is possible that the positive result might origine from impurities rather than from the test susbtance itself. The HPRT assay on a diammonium salt yielded a negative result for mutagenicity in mammalian cells. Therefore, the test substance is considered to be non-genotoxic.


Short description of key information:
In an Ames test conducted according to OECD 471 no genotoxicity was seen. In an in vitro chromosome aberration assay conducted according to OECD guideline 473 an increase in structural chromosome aberrations was seen, probably due to strong precipitation or impurities. An in vivo Micronucleus test according to OECD guideline 475 as well as another chromosome aberration assay in vitro yielded negative results. Based on results of these studies the test substance was considered to be not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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 thirteenth time in Regulation (EU) 2018/1480.