Aluminum cobalt oxide

Endpoint summary

Additional information

The in-vitro genetic toxicity of cobalt aluminium 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 TA1535, TA97a, TA98, TA100, and TA102 at concentrations up to 5000 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. Based on the results of the study, the test substance was not mutagenic in bacteria.

There are no further data available on genetic toxicity for cobalt aluminium oxide. However, there are reliable data for various cobalt and aluminium compounds considered suitable for read-across using the analogue approach. For identifying hazardous properties of cobalt aluminium oxide, the existing forms of the target substance at very acidic and physiological pH conditions are relevant for the assessment of human health effects. As cobalt aluminium oxide is a metal-organic salt, which is insoluble in water at pH 6, it is probable that the target substance has also a low degree of solubility at the physiological pH of 7.4. At acidic pH conditions, however, the study of Stopford et al. (2003) showed that water-insoluble cobalt compounds release cobalt ions. Thus, it can be assumed that cobalt aluminium oxide dissociates at acidic pH in the human body resulting in bioavailable cobalt and aluminate ions. Due to the fact that the toxicological effects of cobalt aluminium oxide are mainly caused by exposure to the cobalt ion, the use of data on soluble cobalt compounds is justified for toxicological endpoints as a worst case scenario. In addition, various aluminium compounds are used within the read-across approach. For further details, please refer to the analogue justification attached in section 13 of the technical dossier.

 

Cobalt compounds

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 (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

 

Aluminium compounds

The study by Oberly et al.(1982) was not considered sufficiently robust to meet this information requirement. Two recent gene-mutation studies conducted by Covance, Inc. (Covance, 2010b) using aluminium chloride (tested up to its solubility limit) and aluminium hydroxide (tested up to 10 mM in suspension and subsequently “cleaned” by Percoll density gradient centrifugation), did not find significant mutations at the thymidine kinase (tk) locus of mouse lymphoma L5187Y cells at any of the doses tested. This study type detects both gene mutations and chromosomal damage.

Covance (2010b)reported negative findings from their assay of forward mutations at thetklocus of L5178Y mouse lymphoma cells. They conducted two experiments with Al(OH)₃, each with a 3 hour treatment duration. Two experiments were also conducted with AlCl₃, one with a 3-hour and the other with a 24-hour treatment duration. Each experiment included incubations with and without metabolic activation. Concentrations of the test items were selected based on results of range-finding studies and observation of precipitation in the incubations. In the Al(OH)₃ experiments, eight concentrations ranging from 6.094 µg/mL to 780 µg/mL were used for determination of mutation frequencies. For AlCl₃, results from five concentrations (from 3.125 to 50 µg/mL) were used in the experiments with 3-hour treatment duration both in the presence and absence of metabolic activation. In the experiments with 24 hour duration treatments, 8 concentrations were used: from 5 to 120 µg/mL in the absence of metabolic activation and from 5 to 50 µg/mL in the presence of metabolic activation. Negative (vehicle) and positive (methyl methane sulphonate with S9 activation; benzo-[a]-pyrene without S9 activation) controls were included in each experiment. The results of the experiments with AlCl₃ were negative: the mutant frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF). A negative linear trend was also observed. In the experiments with Al(OH)₃, the mutation frequencies at all the tested concentrations were less than the sum of the mean control mutant frequency plus GEF. Although a significant positive linear trend in mutation frequencies was observed in the presence of S-9 in one of the experiments with Al(OH)₃, no corresponding increase in mutant frequencies approaching the GEF was observed, and the effect was not observed in the other experiment; therefore, this observation was not considered biologically relevant. This study was conducted in accordance with negative. This study is well described, was conducted in accordance withOECD 476, and with other guidelines, and complied with principles of Good Laboratory Practice.

Oberly et al. (1982) did not observe forward mutations at the thymidine kinase (tk) locus in the L5178Y mouse lymphoma assay with the use of AlCl₃ at concentrations from 2.36 - 2.59 mM.The study was well-described and used a standard assay but did not test a suitable range of concentrations. At least 4 analysable concentrations should be used with the maximum resulting in 10-20% relative survival (OECD 476). The lowest relative survival observed in the study was 38% at 600 µg/mL. A further caveat on the reliability of the results for AlCl₃ is the observation of a large pH change on addition of this substance to the medium, a change that may influence growth. 

The authors report qualitatively that tests with AlCl₃ showed a nonlinear toxic response with, however, little to no increase in mutation frequency. The authors also state that “Other test systems and refinement of test conditions in the mouse lymphoma assay are needed to properly assess the mutagenic potential of this metal”. The results of the study were negative but require qualification as discussed above. It was given a Klimisch Score of 2 but is not considered of adequate quality and reliability to meet the Annex X REACH information requirements for a gene mutation assay in mammalian cells.

The in-vitro micronucleus assay results of Migliore et al. (1999) for aluminium sulphate and the chromosome aberration assay of Lima at el. (2007) using aluminium chloride provide evidence that the aluminium ion is an in-vitro clastogen. Treatment of human lymphocytes with aluminum as AlCl₃has also been observed to induce oxidative DNA damage and inhibit repair of DNA damage from exposure to ionizing radiation (Lankoff et al., 2006). Caicedo et al. (2008), however, did not observe double DNA strand breaks at concentrations up to 5000 µM-Al (as AlCl₃) in human jurkat T-cells, supporting an oxidative mechanism of action that produces single strand effects only. Available studies provide evidence for an indirect genotoxic mechanism of action for the aluminium ion involving the production of single strand breaks. An oxidative mechanism of action would be expected to exhibit a threshold, which may be expected to be higher in-vivo due to more efficient defence mechanisms than in cultured cells.

Thus, there is some evidence that soluble aluminium salts may induce DNA damage, probably by an oxidative mechanism, but these findings were not confirmed in recent GLP studies using the sensitive mouse lymphoma assay. 

 

The most relevant and methodologically strongest in-vivo studies are those conducted by Covance (2010a) and by Balasubramnyam et al. (2009a+b). Covance (2010a) investigated the induction of micronuclei in the bone marrow of rats treated with aluminium hydroxide by oral gavage. This study was conducted in accordance with GLP and recognized testing guidelines. No induction of micronuclei was observed even at the highest dose administered – 2000 mg Al(OH)₃/kg bw/day, two administrations 24 hours apart, equivalent to ca. 690 mg Al/kg bw/day.

Balasubramanyam et al. (2009a+b) examined the genotoxic effects of aluminium oxide particles in vivo. Single doses of aluminium oxide particulate suspensions were administered to rats by oral gavage. The reporting of these investigations was lacking in some areas but the studies appear to have been conducted according to GLP. The study results were positive for the nano-sized materials with evidence of a dose-response relationship. The relevance of these results to the current hazard identification is unclear as it is not distinguishable if the observed effects have arisen from the presence of nanoparticles rather than from any solubilized chemical species (“Al³⁺”) or the chemical substance Al₂O₃ itself. Low toxicity, poorly soluble substances, such as Al₂O₃, when in the form of nanoparticles, have produced inflammatory effects in vitro, possibly due to production of reactive oxygen species (ROS) (Duffin et al., 2007; Dey et al., 2008). Current scientific knowledge does not allow the distinguishing of genotoxic effects due to the physical (in this case “nanoparticle”) nature of the exposure from genotoxic effects due to the chemical characteristics of the substance (Landsiedel et al., 2009; Singh et al., 2009; Gonzalez et al., 2008). However, in the current extensive debate concerning the genotoxic effects of nanoparticles of many different substances, the possibility that nanoparticles stimulate an inflammatory response which leads to oxidative stress and thence to DNA damage has been widely voiced. The genotoxicity levels for 50 to 200μm diameter particles (Al₂O₃-bulk) were also not statistically significantly different from those for the control. Balasubramanyam et al. (2009a+b) reported tissue aluminium oxide levels elevated in a dose-response manner for the groups treated with nano-sized materials, consistent with transfer of the nano-sized particles across the gastrointestinal mucosa (Florence, 1997; Hagens et al., 2007). A particle size dependence of gastrointestinal absorption was apparent. Aluminium oxide levels in the tissues of animals dosed with the larger 50 to 200 μm diameter particles (Al₂O₃-bulk) were not elevated to a statistically significant level. 

Short description of key information:
Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimuriumTA1535, TA97a, TA98, TA100, and TA102: negative with and without metabolic activation (according to OECD 471)
Read-across with cobalt compounds:
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 aluminium compounds:
For aluminium compounds, there is limited evidence for genetic toxicity in in-vitro and in-vivo experiments, but not sufficient for classification.
Based on a worst case scenario, there is sufficient evidence to classify cobalt aluminium oxide as genotoxic due to data on soluble cobalt compounds.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

Based on the 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.