Reaction mass of disodium [2,2'-(imino-kappaN)dibutanedioato-kappa2O1,O4(4-)]manganese(2-) and sodium sulphate

Endpoint summary

Additional information

No genetic toxicity studies are available for the target substance Mn (2Na) IDHA. Therefore, the data on free IDHA chelating agent and the structurally similar Mn(2Na)EDTA have been used to assess genetic toxicity potential of Mn(2Na)IDHA (please refer to read-across statement). Additionally, the results of in vitro and in vivo tests with inorganic manganese compounds have been taken into account since the systemic toxicity of Mn(2Na)IDHA can also be mediated by manganese released from complexes.

Mutagenicity in bacterial cells with Mn(2Na)EDTA

The test substance, EDTA-MnNa2, was examined for mutagenic activity in the bacterial reverse mutation test using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan requiring Escherichia coli strain WP2 uvrA, in the absence and presence of a liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix) (OECD 471; Wijngaard van de, 2009; Report No. V8405/14). The test substance was dissolved in phosphate buffered saline (PBS). One bacterial reverse mutation test was performed. All strains were used, in the absence and presence of S9-mix, with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (PBS) and positive controls were run simultaneously with the test substance. The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test was considered valid. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In both the absence and presence of S9-mix in all strains, EDTA-MnNa2 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that EDTA-MnNa2 is not mutagenic under the conditions employed in this study.

In vitro Micronucleus test with Mn(2Na)EDTA in human lymphocytes

The test substance EDTA-MnNa2 was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes, in both the absence and presence of a metabolic activation system (S9-mix) with duplicate cultures (OECD 487; Vogel de, 2009; Report No. V8417/06). Two separate in vitro micronucleus tests were conducted for which blood was obtained from two different donors. In the first test, in the presence and absence of S9 -mix, the treatment/recovery time was 4/20 hours (pulse treatment method). In the second test, concentration spacing was modified and the treatment/recovery times were 20/28 hours (continuous treatment) in the absence of S9-mix. Dose levels, ranging from 7.8 to 3891 µg/mL, were tested. In all instances, the maximum final concentration in the culture medium was 10 mmol/L. A purity of 92.3% was taken into account while preparing the dosing solutions. Culture medium was used as solvent for the test substance. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). Cyclophosphamide, a clastogenic compound which requires metabolic activation, was used as positive control in the presence of S9-mix. A known clastogenic compound (Mitomycin C) and a known aneugenic compound (Vinblastine sulphate) were used as positive controls in the absence of S9-mix.

In the first test, in the presence and absence of S9-mix, the highest dose level (3891 µg/mL) was cytotoxic to the cells based on the CBPI. Analysis of micronuclei formation was carried out in the cultures of three dose levels (1000, 2000 and 3891 µg/mL) of the test substance, the cultures of the solvent control and the cultures of the positive controls. In the first test, the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei.

In the second test, the two highest dose levels (3000 and 3891 pg/mL) were cytotoxic to the cells based on the CBPI. Analysis of micronuclei formation was carried out in the cultures of three dose levels (2500, 3000 and 3891 µg/ml) of the test substance, the cultures of the solvent control and the cultures of the positive controls. In the second test, the test substance did not induce a statistically significant increase in the number of binucleated cells containing micronuclei.

Treatment with the positive controls Cyclophosphamide, Vinblastine sulphate and Mitomycin C resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the solvent control. This demonstrates the validity of the study. From the results obtained in two in vitro micronucleus tests it is concluded that, under the conditions used in this study, the test substance EDTA-MnNa2 was neither clastogenic nor aneugenic to cultured human lymphocytes.

Mutagenicity in bacterial cells with the chelating agent IDHA

IDHA was investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants (OECD 471; Herbold, 1997a). These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. In a first experiment, doses up to and including 1581 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness of this effect this range could nevertheless be used for assessment purposes. Moreover, IDS, Na-Salz was investigated in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per tube after preincubation for 20 minutes at 37°C on the same Salmonella typhimurium LT2 mutants. In this experiment, doses up to and including 5000 µg per tube did not cause any bacteriotoxic effects: total bacteria counts remained unchanged and no inhibition of growth was observed.

In both experiment, evidence of mutagenic activity of IDHA was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. Therefore, IDHA was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

In vivo Micronucleus Test in mice with the chelating agent IDHA

The micronucleus test was employed to investigate IDHA (CAS 144538 -83 -0) in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts (OECD 474; Herbold, 1997b). The known clastogen and cytostatic agent, cyclophosphamide, served as positive control. The treated animals received a single intraperitoneal administration of IDHA or cyclophosphamide. The femoral marrow of groups treated with IDHA was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of IDHA and the positive control, cyclophosphamide, were 1500 and 20 mg/kg body weight, respectively.

The animals treated with IDHA showed symptoms of toxicity after administration. Four of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 1500 mg/kg IDHA. There was an altered ratio between polychromatic and normochromatic erythrocytes. However, the frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint and the number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can only be used as the endpoint of the assay when animals are treated continuously for 4 weeks or more. The increase in micronucleated polychromatic erythrocytes, due, for example, to chromosome breaks or spindle disorders, is the criterion for clastogenic effects in this test model. The results with IDHA gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 1500 mg/kg. Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with miconuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

In conclusion, there was no indication of a clastogenic effect of an intraperitoneal dose of 1500 mg/kg IDHA in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

In vivo Commet Assay with the chelating agent IDHA

IDHA was tested for genotoxic activity in the in vivo comet assay after single oral treatment of male Wistar rats with doses of 2500 mg/kg and 5000 mg/kg (Brendler-Schwaab, 2001). Ethyl-methanesulfonate (EMS) served as positive control at a dose of 200 mg/kg. Hepatocytes and kidney cells were prepared 3 hours after administration. Animals treated with 5000 mg/kg IDHA showed minor symptoms of toxicity, namely roughened fur. No cytotoxicity was observed in isolated cells. No biologically relevant increase of the tail length were observed in hepatocytes and kidney cells after single oral treatment of male rats with IDHA in doses of up to 5000 mg/kg compared to the negative control animals. The positive control EMS had clear genotoxic effects in hepatocytes as well as in kidney cells as shown by the biologically relevant increase of the tail length, demonstrating the sensitivity of the method used for the detection of induced DNA damage. Based on these results and under the conditions described, IDHA was considered to be non-genotoxic in the in vivo comet assay in hepatocytes and kidney cells of male rats.

Results of in vitro and in vivo tests with inorganic manganese compounds

Manganese is distributed throughout all cells in the body; therefore, it is present in germ cells. However, existing studies in humans and animals are not sufficient to predict if distribution of excess manganese into germ cells might result in heritable genetic changes. Manganese is constantly present in human tissues and, therefore, is able to enter germ cells. One human study involving inhalation exposure to nickel and manganese observed chromosomal aberrations in welders working with these metals (Elias et al. 1989, cited in ATSDR, 2012). However, the presence of nickel is a confounding factor, as it is known for causing chromosomal changes.

In vivo assays in mice showed that oral doses of manganese sulfate or potassium permanganate caused micronuclei and chromosomal aberrations in bone marrow (Joardar and Sharma 1990, cited in ATSDR, 2012). In contrast, oral doses of manganese chloride did not cause chromosomal aberrations in the bone marrow or spermatogonia of rats (Dikshith and Chandra 1978, cited in ATSDR, 2012).

Studies in animals are equivocal; there are not enough data to make predictions as to the likelihood for excess exposures of manganese to cause heritable genetic changes. The results of in vitro studies are also conflicting. While manganese sulfate was shown to not be mutagenic to Salmonella typhimurium strains TA97, TA98, TA100, TA1535, or TA1537 in a study, it was shown to be mutagenic to strain TA97 in another study. Manganese chloride was reportedly not mutagenic in S. typhimurium strains TA98, TA100, and TA1535, but it was mutagenic in strain TA1537, and conflicting results were obtained for TA102. In vitro assays in mammalian cells manganese chloride produced gene mutations in cultured mouse lymphoma cells and caused DNA damage in vitro human lymphocytes (depending on concentration). Manganese sulfate induced sister chromatic exchange in Chinese hamster ovary (CHO) cells at only G2 phase of cell cycle but it was not clastogenic in FM3A cells. Similar results were obtained with potassium permanganate.

The results of in vitro studies show that at least some chemical forms of manganese have mutagenic potential. However, as the results of in vivo studies in mammals are inconsistent, no overall conclusion can be made about the possible genotoxic hazard to humans from exposure to manganese compounds. Data on carcinogenicity, mutagenicity and genotoxicity are inconclusive and inadequate to establish a definitive position on the carcinogenicity of manganese and its compounds (SCOEL, 2011).

Justification for selection of genetic toxicity endpoint
No study is selected since the studies are all weight-of-evidence pieces.

Short description of key information:
1) Ames test with Mn(2Na)EDTA (OECD 471): Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100; Escherichia coli strain WP2 uvr; negative with and without metabolic activation;
2) In vitro MNT (human lymphocytes) with Mn(2Na)EDTA (OECD 487): neither clastogenic nor aneugenic;
3) Ames test with IDHA (OECD 471): Salmonella typhimurium strains A 1535, TA 100, TA 1537, TA 98 and TA 102; negative (preincubation and plate incorporation methods);
4) In vivo MNT with IDHA (bone marrow erythrocytes) (OECD 474): negative;
5) In vivo Commet Assay in rats with IDHA; negative in hepatocytes and kidney cells of male rats;
6) ATSDR (2012) and SCOEL (2011) data on genetic toxicity of Inorganic manganese compounds: numerous in vitro and in vivo studies (inconclusive and inadequate result).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The read-across substance Mn(2Na)EDTA was negative in the Ames test and in in vitro Micronucleus test in human lymphocytes. The other read-across substance chelating agent IDHA was also negative in the available tests: Ames test (pre-incubation and plate incorporation methods), in vivo Micronucleus Test in bone marrow erythrocytes and in vivo Commet Assay. Regarding genetic toxicity potential of manganese, no definitive conclusion can be made since the data on mutagenicity and genotoxicity are inconclusive and inadequate. Due to the inert nature of the chelates as a group, their low chemical reactivity and high stability constant (in opposite to the inorganic manganese salt compounds which rather dissociate in aquatic environments or body fluids) no genetic toxicity potential can be attributed to Mn(2Na)IDHA. As overall conclusion, Mn(2Na)IDHA does not meet the criteria for classification and labelling for genetic toxicity end point in accordance with European regulation (EC) No. 1272/2008.