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

Summary of genetic toxicity test results available for Cu(2Na)EDTA

Cu (2Na) EDTA was negative in the Ames test (OECD 471; BASF, 1992; Report No. 40M0401/914239) and in the in vitro Micronucleus test (OECD 487, Usta, 2013; Report No. V20217/03) using a treatment period of 4 h (with and without S9 -mix). In the in vitro Micronucleus test using a treatment period of 20 h (continuous treatment without S9 -mix), EDTA-CuNa2 was positive at levels >= 62.5 µg/mL, inducing aneugenic but no clastogenic effects. This long treatment period together with the high concentrations of chelate may have resulted in exchange and substantial binding of essential elements such as zinc. Similar results were obtained with EDTA-FeNa and DTPA-FeNaH (described in RAR, 2004). Heimbach et al (2000) concluded that the lack of effects by the Zn-EDTA salt in contrast to effects induced by Ca2+, Na+ and Mn2+ salts of EDTA, provided evidence that zinc is required for the initiation or continuation of DNA synthesis and maintaining cell function. As such, the significance of mutations produced by Cu (2Na) EDTA (and also EDTA-FeNa and DTPA-FeNaH) at non-physiological concentrations in an in vitro screening system is difficult to extrapolate for relevance to intact organisms. Although no in vivo genotoxicity studies have been carried out with Cu (2Na)EDTA, several in vivo genotoxicity studies are available for other EDTA-compounds such as EDTA-Na2H2 (RAR, 2004). No genotoxic activity was observed. Therefore, the overall finding for EDTA chelates is that they lack significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function.

Mutagenicity in bacterial cells with the chelating agent IDHA

IDS, Na-salt 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 (Herbold, B, 1997a, OECD 471). 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-salt 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 IDS, Na-salt 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, IDS, Na-salt 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

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

The animals treated with IDS, Na-salt 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 IDS, Na-salt. 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 IDS, Na-salt 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 IDS, Na-salt 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

IDS, Na-salt 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, S., 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 IDS, Na-salt 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 IDS, Na-salt 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, IDS, Na-salt 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 copper compounds

SCOEL, 2013:

Copper compounds were negative in most studies in bacteria and yeasts but positive in mammalian cells. There were dose-dependent increases in gene mutations, sister chromatid exchange, DNA strand breaks and unscheduled DNA synthesis. Besides this, oxidative DNA base modifications, interaction with the repair of oxidative DNA damage and inhibition of poly(ADP-ribosyl)ation were observed in different cells and cell lines (V79 hamster cells, rat hepatocytes, CHO cells, human fibroblasts, HeLa cells, human CD4+ T cells, and peripheral mouse blood lymphocytes).

Copper sulphate compounds (degree of hydratation is not specified) induced chromosomal aberrations, breaks and increase in micronuclei in mice of different strains. On contrary, copper sulphate pentahydrate induced an increase in chromosomal aberrations (chromatid type) and chromosomal breaks in Albino mice treated intraperitoneally, while no increase in micronuclei following a single intraperitoneal injection was observed in CBA mice.

 

ATSDR, 2004:

Several studies on copper sulfate and copper chloride genotoxicity did not find significant increases in the occurrence of reverse mutations in Salmonella typhimurium or Saccharomyces cerevisiae. In contrast, an increased occurrence of reverse mutations in Escherichia coli was found. Positive results have been found in studies testing for DNA damage: errors in DNA synthesis by viral DNA polymerase, a reduction in DNA synthesis and an increase in the occurrence of DNA strand breaks. The increase in sister chromatid exchange in Chinese hamster cells is consistent with the clastogenic effects observed in in vivo assays.

No studies were located regarding genotoxicity in humans after inhalation, oral, or dermal exposure to copper or its compounds. Several studies have assessed the genotoxicity of copper sulfate following oral or parenteral exposure; the results of these in vivo genotoxicity studies are summarized in Table 3-3 (attached to this file). Significant increases in the occurrence of micronuclei and chromosomal aberrations have been observed in chick bone marrow cells and erythrocytes (Bhunya and Jena 1996, cited in ATSDR, 2004) and mouse bone marrow cells (Agarwal et al., 1990; Bhunya and Pati 1987, cited in ATSDR, 2004). A study by Tinswell and Ashby (1990) did not find increases in the number of micronuclei in mouse bone marrow cells. Increases in the occurrence of recessive lethals (Law, 1938, cited in ATSDR) and sperm abnormalities (Bhunya and Pati 1987, cited in ATSDR, 2004) have also been observed in Drosophila and mice, respectively.


Justification for selection of genetic toxicity endpoint
No study is selected since all studies are negative.

Short description of key information:
1) Ames test with Cu(2Na)EDTA (OECD 471): Salmonella typhimurium strains TA 1537, TA 1538, TA 98, TA 100; Escherichia coli strain WP2 uvr; negative with and without metabolic activation;
2) In vitro MNT (human lymphocytes) with Cu(2Na)EDTA (OECD 487): neither clastogenic nor aneugenic;
3) Review of study results of other EDTA chelates (Heimbach et al.,, 2000): EDTA chelates are not genotoxic (overall conclusion)
4) 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);
5) In vivo MNT with IDHA (bone marrow erythrocytes) (OECD 474): negative;
6) In vivo Commet Assay in rats with IDHA; negative in hepatocytes and kidney cells of male rats;
7) SCOEL (2013) and ATSDR (2004) data on genotoxicity of inorganic copper compounds: copper compounds were negative in most studies in bacteria and yeasts but positive in in vitro mammalian cells; ambigous result in in vivo studies: copper sulphate (degree of hydratation is not specified) induced chromosomal aberrations, breaks and increase in micronuclei in mice of different strains, while inconsistent data are available on copper sulphate pentahydrate.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the large toxicity data available for inorganic copper salts and copper chelates, the overall evidence is that toxicity observed in animal studies is triggered by high doses of copper metal cation. Binding to biomolecules, copper modulates, changes or inactivates a lot of molecular cascades on the cellular, tissue-, organ or organism’s level. However, the chemistry and the reactivity of inorganic copper salts are totally different from those of the chelates. Whereas inorganic copper salts dissociate almost complete to ions, chelates hold copper making it immobile in both aquatic environments and body fluids. Hence, the reactivity of the chelated copper is much less. Moreover, copper ions can be freely available for absorption in case of ingestion, they can be absorbed through cell membranes by passive transport (ion channels or pores), while chelates are large molecules and need rather an active transport system to be absorbed. In this regard, not only the availability of copper but also its absorption rates will be much lower in case of chelates therewith lowering the toxicity significantly. This has been proved by the numerous long-term toxicity studies in animals with CuEDTA and copper inorganic salts like copper sulfate (SCOEL, ATSDR references).

In in vitro genetic toxicity studies, inorganic copper compounds were negative in most studies in bacteria and yeasts but positive inin vitromammalian cells. Ambiguous result was obtained in in vivo studies: copper sulphate (degree of hydratation is not specified) induced chromosomal aberrations, breaks and increase in micronuclei in mice of different strains, while inconsistent data are available on copper sulphate pentahydrate. On the contrary, the chelated copper, viz. the read-across substance Cu(2Na) EDTA gave negative results in two in vitro mutagenicity studies: in the Ames test and the micronucleus test following exposure for 4 h (with and without S9 mix). The positive result (aneugenicity but not clastogenicity) was only observed following exposure for 20 h (without S9 -mix). Such an outcome can be explained by Zn deficiency caused by Cu(2Na)EDTA, especially expected under conditions without S9 mix . The Zn depletion mentioned in the review of Heimbach et al. (2010) is a possible explanation for perturbation in DNA synthesis and positive outcomes of some mutagenicity tests of the chelated EDTA compounds. The overall conclusion, however, is that “EDTA-metal complexes lack significant genotoxic potential under conditions that do not deplete essential trace elements required for normal cell function” (Heimbach et al., 2010). Therefore, the positive genetic toxicity potential of inorganic copper compounds cannot be directly assigned to chelated copper compounds.

The other read-across substance chelating agent IDHA was 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. These results additionally confirm that genotoxicity (as well as general systemic toxicity in animal studies) is driven by copper and not by the chelating agent IDHA.

In conclusion, the inert nature of the chelates as a group, their low chemical reactivity and high stability constant (in opposite to the inorganic copper salt compounds which rather dissociate in aquatic environments and body fluids) no genetic toxicity potential can be attributed to Cu(2Na) IDHA.

As overall conclusion, Cu (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.