Cobalt molybdate

Endpoint summary

Additional information

The in-vitro genetic toxicity of cobalt molybdenum oxide was investigated in a bacterial reverse mutation assay (Ames test) according to OECD 471 (Andres, 2012). The plate incorporation and the pre-incubation method was conducted with S. typhimurium strains TA 1535, TA97a, TA98, TA100, and TA102 at concentrations up to 150 µg/plate. The test substance did not induce reversions in the tested strains with or without metabolic activation. No cytotoxic effects were observed and all positive controls were valid. As no complete dissolution of the test substance in the vehicle (water) was possible, undissolved particles were visible on the plates.

There are no further data available on genetic toxicity for cobalt molybdenum oxide. However, there are reliable data for soluble cobalt and molybdenum substances considered suitable for read-across using the analogue approach. For identifying hazardous properties of cobalt molybdenum oxide, the existing forms of cobalt molybdenum oxide at very acidic and physiological pH conditions are relevant for risk assessment of human health effects. Cobalt molybdenum oxide is a metal-organic salt, which is highly water soluble (~ 508 mg/L) and nearly completely dissociates in aqueous solutions. As it is expected that cobalt molybdenum oxide is capable of forming ions at very acidic and physiological pH conditions, cobalt cations and molybdate anions will be present and completely bioavailable, same as for other soluble cobalt and molybdenum compounds.Due to the existing cobalt and molybdate ions, data from other soluble cobalt and molybdenum substances are used in the derivation of toxicological endpoints for cobalt molybdenum oxide. For further details refer to the analogue justification.

 

Soluble cobalt substances

Soluble cobalt(II) salts were found to be primarily negative in Salmonella typhimurium strains with and without activation (Zeiger et al., 1992; Ogawa et al., 1986; Tso and Fung, 1981). However, Ogawa et al. (1986) could show that when the Ames test was performed with 9-aminoacridine in the presence of varying concentrations of cobalt(II)chloride, a remarkable increase in the mutagenic activity was observed in TA1537 and TA2637. Furthermore cobalt(II)chloride could induce reversions when combined with 4 -aminoquinoline, harman or norharman, which were not themselves mutagenic in the strains TA1537 or TA2637. In mammalian cells, mutagenicity and cell transformation assays gave mixed results. However, cobalt chloride induced DNA damage (strand breaks and DNA-protein cross links), chromosome damage (micronuclei and sister chromatid exchanges) and aneuploidy in in-vitro experiments (Kitahara et al., 1996; 1980; Hartwig et al., 1990, Miyaki et al., 1979; McLean et al., 1982; Hamilton-Koch et al., 1986; Ponti et al., 2009, Kasten et al., 1997; Doran et al., 1998). In mouse bone marrow cells, micronucleus formation was not significantly altered by treatment with cobalt(II)chloride hexahydrate in the presence or absence of S9 mix (Suzuki et al., 1993). However, no positive control substance was used to validate this assay.

In-vivo, a single intraperitoneal injection of cobalt chloride hexahydrate induced micronuclei in mouse bone marrow (Suzuki et al., 1993). 30 hours following the injection of the test substance in mice, a dose-dependent increase in micronucleus formation was seen at 90 and 50 mg/kg bw, but not at 25 mg/kg bw (equivalent to 22.3, 12.4 and 6.2 mg/kg bw cobalt molybdenum oxide) (Suzuki et al., 1993). Cobalt chloride also induced aneuploidy, pseudoploidy and hyperploidy in the bone marrow and testes of hamsters when dosed intraperitoneally over 9 days (Farah, 1983). In mice, chromosome aberrations in the bone marrow were observed after single oral treatment with cobalt chloride (Palit et al., 1991a, b, c, d). There was no indication of increased DNA strand breaks or micronuclei in blood lymphocytes of 35 workers in a cobalt refinery exposed to cobalt dust compared to 27 unexposed workers (DeBoeck et al., 2000).

Taking into account all available data, in-vitro and in-vivo genotoxicity data indicated that soluble cobalt substances can cause DNA and chromosome damage. However, these effects are likely mediated by indirect mechanisms including the generation of reactive oxygen species, increased oxidative stress and inhibition of DNA repair enzymes (Asmuss et al., 2000, Kopera et al., 2004; Snyder et al., 1989; Kasten et al., 1997).

 

Reference not cited in the IUCLID:

Asmuss M, Mullenders LH, Hartwig A. 2000. Interference by toxic metal compounds with isolated zinc finger DNA repair proteins. Toxicology Letters, 112–113:227–231

Farah, SB. 1983, The in vivo effect of cobalt chloride on chromosomes. Rev. Brasil. Genet. 6:433–442

Doran et al., 1998, Neoplastic transformation of cells by soluble but not particulate forms of metals used in orthopaedic implants. Biomaterials. 19: 751–759

Hamilton-Koch et al., 1986, Metal-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells. Chem.-biol. Interact. 59: 17–28

Hartwig et al., 1990, Uptake and genotoxicity of micromolar concentrations of cobalt chloride in mammalian cells. Toxicol. environ. Chem. 28: 205–215

Kasten U, Mullenders LH, Hartwig A. 1997.Cobalt(II) inhibits the incision and the polymerization step of nucleotide excision repair in human fibroblasts.Mutat Res 383:81-90

Kitahara et al., 1996, Mutagenicity of cobalt and reactive oxygen producers.Mutat. Res. 370: 133–140

Kopera E, Schwerdtle T, Hartwig A, Bal W. 2004.Co(II) and Cd(II) Substitute for Zn(II) in the Zinc Finger Derived from the DNA Repair Protein XPA, Demonstrating a Variety of Potential Mechanisms of Toxicity. Chem. Res. Toxicol. 17:1452-1458

McLean et al., 1982, Rapid detection of DNA strand breaks in human peripheral blood cells and animal organs following treatment with physical and chemical agents. In: Bora, K.C., Douglas, G.R. & Nestmann, E.R., eds, Progress in Mutation Research, Vol. 3, Amsterdam, Elsevier Biomedical Press, pp. 137–141

Miyaki et al., 1979, Mutagenicity of metal cations in cultured cells from Chinese hamster. Mutat. Res. 68: 259–263

Palit et al., 1991a, Modification of the clastogenic effects of cobalt by calcium in bone marrow cells of mice in vivo. Cytologia 56:373-377

Palit et al., 1991b, Chromosomal aberrations induced by cobaltous chloride in mice in vivo. Biol Trace Elem Res 29:139-145

Palit et al., 1991c, Cytotoxic effects of cobalt chloride on mouse bone marrow cells in vivo. Cytobios 65:85-89

Palit et al., 1991d, Protection by chlorophyllin against induction of chromosomal aberrations by cobalt in bone marrow cells of mice in vivo. Fitoterapia 62:(5)425-428

Tso, WW and Fung, WP, 1981, Mutagenicity of metallic cations. Toxicology Letters, 8: 195 -200

Ponti J, Sabbioni E, Munaro B, Broggi F, Marmorato P, Franchini F, Colognato R, Rossi F. 2009. Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3T3 mouse fibroblasts. Mutagenesis. 24: 439 - 445.

Snyder, R.D., Davis, G.F., Lachmann, P.J. 1989. Inhibition by metals of X-ray and ultraviolet induced DNA repair in human cells. Biol. trace Elem. Res. 21: 389–398

 

Molybdenum substances

Three recently conducted guideline conform, highly reliable state-of-the-art genotoxicity tests produced unequivocally negative results, and thus provide strong evidence for an absence of concern for genotoxic effects of molybdenum substances, which is supported by reliable supplementary data.

The following recently conducted guideline-conform, highly reliable state-of-the-art genotoxicity tests with sodium molybdate are available:

- an bacterial reverse mutation assay (Beevers, 2009),

- an in-vitro micronucleus assay in human lymphocytes (Fox, 2005; Taylor, 2009), and

- an in-vitro gene mutation assay (tk) in mouse lymphoma cells (Lloyd, 2009).

All three tests produced unequivocally negative results, and thus provide strong evidence for an absence of concern for genotoxic effects of molybdenum substances.

Apart from these reliable test results, a large number of published studies exist of varying quality and extent of documentation, which is why these were subjected to a detailed quality and reliability screening, the outcome of which can be summarised as follows:

(i) unequivocally negative results were obtained in guideline-conform, valid bacterial reverse mutation assays for high purity (moderately soluble) molybdenum trioxide (Jones, 2004 and Zeiger et al., 1992). Negative results were also obtained in the same test system with (highly soluble) sodium and ammonium molybdates, which however from a perspective of formal regulatory compliance are considered incomplete because of the selected tester strains (Armitage, 1997).

(ii) all in-vitro clastogenicity test considered as assignable and reliable with or without restrictions are negative (Fox, 2005).

(iii) Available in-vivo studies were subjected to a thorough evaluation and were found to be seriously flawed and of poor quality, and were thus assigned reliability grades of either 3 or 4. In this context it is explicitly noted that set of studies published by Titenko-Holland (1998) have already previously been criticised and disregarded by the Classification and Labelling Committee of the ECB as deficient during discussions on molybdenum trioxide in 2004.

 

References not cited in the IUCLID:

Armitage, A. (1997). Ammonium octamolybdate-Salmonella plate incorporation mutagenicity assay (Ames test) with strains TA98, TA 100, TA 1535 and TA 1537. Testing laboratory: Springborn Laboratories, INC., Health and Environmental Sciences, 790 main Street, Report no.: 97-4-6931

Beevers, C. (2009). Reverse mutation in five histidine-requiring strains of Salmonella typhimurium. Testing laboratory: Covance Laboratories Ltd., North Yorkshire HG3 1PY, ENGLAND. Report no.: 2992/1. Owner company: International Molybdenum Association.

Jones, E. (2004). Sublimed undensified pure molybdenum trioxide: Bacterial mutation assay in S. typhimurium and E. coli. Testing laboratory: Central Toxicology Laboratory, Alderley Park Macclesfield. Report no.: YV6553. Owner company: International Molybdenum Association,.Report date: 2004-04-01.

Titenko-Holland, N et al.(1998). Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo. Environmental and Molecular Mutagenesis 32: 251-259

Titenko-Holland, N. et al. (1998a). Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo. Environmental and Molecular Mutagenesis 32: 251-259

Titenko-Holland, N. et al. (1998b). Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo. Environmental and Molecular Mutagenesis 32: 251-259

Zeiger, E. et al. (1992). Salmonelly Mutagenicity Tests: V. Results from the Testing of 311 Chemicals.Environmental and Molecular Mutagenesis Volume 19, Supplement 21:2 - 141

Short description of key information:
Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimuriumTA 1535, TA97a, TA98, TA100, and TA102: negative with and without metabolic activation (according to OECD 471)
 
Read-across with soluble cobalt substances:
In vitro: Mutagenicity assays in bacteria with soluble cobalt(II)salts were primarily negative with and without metabolic activation. However, cobalt(II)salts induced DNA damage (strand breaks and DNA-protein cross links), chromosome damage (micronuclei and sister chromatid exchanges) and aneuploidy.
In vivo: Cobalt(II)salts induced chromosome damage (micronuclei), aneuploidy, pseudoploidy and hyperploidy.
 
Read-across with soluble molybdenum substances:
In-vitro: A bacterial reverse mutation assay, a micronucleus assay in human lymphocytes and a gene mutation assay (tk) in mouse lymphoma cells showed negative results.
 
As positive results in genetic toxicity tests in-vitro and in-vivo are mainly attributed to the bioavailable cobalt ion, cobalt molybdenum oxide is also considered to be genotoxic.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

Based on an analogue approach, the available data on genetic toxicity meet the criteria for classification as Category 2 (H341) according to Regulation (EC) 1272/2008 and as R68 (Category 3) according to Directive 67/548/EEC.