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There are no studies available for “Reaction product of thermal process between 1000°C and 2000°C of mainly aluminium oxide and calcium oxide based raw materials with at least CaO+Al2O3+MgO >80% , in which aluminium oxide, magnesium oxide and calcium oxide in varying amounts are combined in various proportions into a multiphase crystalline matrix”. As this substance is an UVCB substance with aluminium oxide (Al2O3), calcium oxide (CaO) and magnesium oxide (MgO) as main constituents and magnesium oxide (MgO) is exempted from registration according to EC 1907/2006 Annex V Section 10, data from aluminium and calcium compounds were taken into account by read across following a structural analogue approach.

Read-across from aluminium compounds:

Information available on the genotoxic potential of aluminium compounds was taken into account for hazard assessment, since the pathways leading to toxic outcomes are considered to be dominated by the chemistry and biochemistry of the aluminium ion (Al3+) (Krewski et al., 2007; ATSDR, 2008).

 

Bacterial test systems

Bacterial mutagenicity assays of simple aluminium compounds have been negative in bacterial reverse mutation tests using several strains of S. typhimurium (e. g.; Marzin and Phi, 1985 (TA102); Ahn and Jeffrey, 1994 (TA98); Gava et al., 1989 (TA92, TA98, TA100, TA104); Blevins and Taylor, 1982 (Spot Test: TA98, TA100, TA1535, TA1537, TA1538); Pan et al., 2010 (TA97a, TA100)) and Escherichia coli WP2 trp uvrA (Pan et al., 2010). Results from the Rec Assay using Bacillus subtilis H17 (Rec+, arg-, tryp-) and B. subtilis M45 (Rec-, arg-, tryp-) have also been negative (Nishioka et al., 1975; Kanematsu et al., 1980).  

As uptake of aluminium depends on the chemical form of the aluminium substance, particularly its solubility and other ligands present, the available mutagenicity assays have tended to use aluminium(III) salts that are more soluble than the target substances. It should be noted that some strains of bacteria are sensitive to only certain types of genetic changes (Battersby et al., 2007) requiring the use of, for example, a range of S. typhimurium strains to provide suitable sensitivity. Strains TA102 and TA104 are sensitive to oxidising mutagens. Given the possibility of false negatives due to the exclusion of Al3+ion by cell membranes, and possible insensitivity of assays to mechanisms of metal genotoxicity e. g. induction of large DNA deletions which would lead to cell death rather than mutation (see also Battersby et al., 2007), negative results from bacterial test systems, although contributing to the weight of evidence, are not sufficient to negate further testing.

Animal Studies –In-vivo Somatic Cell Tests

The most relevant and methodologically strongest 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 (Klimisch Score=1). 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 the observed effects mayhave arisen from the presence of nanoparticles as foreign bodies in the cells 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 thenano-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 200μm diameter particles (Al2O3-bulk) were not elevated to a statistically significant level. 

The positive results observed in studies of Aluminium sulphate reported by Dhir et al. (1990) and Roy et al. (1992) occurred with non-physiologically-relevant intraperitoneal administration of the test substances and were methodologically weaker (Klimisch Scores of 2-3).

Thus, on weight of evidence, aluminium compounds of normal particle size (i. e. not nanoparticles) do not induce genotoxic effects in somatic cellsin-vivowhen administered by a physiologically relevant route.

Animal Studies –In-vivo Germ Cell Tests

Results from animals’ studies that have employed the dominant lethal assay are inconsistent. Guo et al. (2005) (Klimisch Score=3) reported a positive response in rats subcutaneously dosed with AlCl3 daily (0, 7 and 13 mg Al/kg bw/day) for a two week period prior to 9 weeks of sequential matings. Two other studies (Dixon et al., 1979; Zelic et al., 1998), both of which were also assigned Klimisch Scores of 3, reported negative results, but the assessments of dominant lethal effects were very limited. Only Guo et al. (2005) reported aluminium levels in the testes indicating that aluminium had reached the target tissue. However, confidence in these results is limited by the high serum Al concentrations in the control group, the time delay in effect, the lack of information on the mating schedule and number of females, and the concurrent effects on fertility (fecundity, libido and male gamete histology). The evidence from animal studies for a mutagenic hazard to germ cells from aluminium ion is inconsistentand no clear conclusions about germ cell mutagenicity can be made based on these studies.

Since there are no known examples of substances inducing genotoxic effects in germ cells that are not genotoxic in somatic cells, it is highly unlikely that aluminium compounds pose any genotoxic risk to germ cells.

In-Vitro Gene Mutation Assays

The study by Oberly et al. (1982) (Klimisch Score=2) 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. These studies were conducted according to guidelines and GLP (Klimisch =1 for Aluminium chloride and 2 for Aluminium hydroxide).

In-Vitro Mammalian Cell Assays

The in-vitro micronucleus assay results of Migliore et al. (1999) for aluminium sulphate and the chromosome aberration assay of Lima et al.(2007) using Aluminium chloride provide evidence that the aluminium ion is an in-vitro clastogen. Treatment of human lymphocytes with aluminum as AlCl3 has 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 lymphomatkassay. 

Human Studies

Two human studies, Botta et al. (2006) and Iarmarcovai et al. (2005) became available following the completion of the ATSDR (2008) and Krewski et al. (2007) reviews of aluminium. However, these studies contribute little to the weight of evidence approach for assessing the mutagenic potential of the target substances as the results are confounded by the complex nature of the exposure (a mixture of fume from different welding materials), possible co-exposures, and uncertainties concerning the completeness of the adjustments for age and smoking.

Other Relevant Information

In a weight of evidence assessment for a mutagenic effect in humans, the levels at which effects are seen in animal studies and the systemic bioavailability of the target substances need to be considered. The study conducted by Covance (2010b) in non-fasted rats observed no induction of micronuclei in bone marrow at the acute maximum tolerated dose (MTD) for Aluminium hydroxide when administered by oral gavage, namely 2000 mg Al(OH)3/kg bw/day. The MTD had been determined previously in a range-finding experiment. Balasubramanyam et al. (2009a) also observed no statistically significant genotoxic effects in rat bone marrow after a single oral gavage of 2000 mg Al2O3/kg bw in the form of particles with a size-range of 50 to 200 µm. Although current toxicokinetic information does not allow the prediction of time profiles of levels of aluminium in target tissues as a function of realistic external exposures, when administered orally or by inhalation the target substances exhibit low bioavailability.

 

Read-across from calcium compounds:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the Escherichia coli strain WP2 uvrA were treated with the test material PRECAL 30S (Calcium oxide (burnt lime)) diluted in 1M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES) using the Ames plate incorporation method according to OECD Guideline No. 471. The substance was tested up to 2500 µg/plate with and without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the strains, with any dose of the test material, either with or without metabolic activation (Sokolowski, 2007).

 

In addition, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the Escherichia coli strain WP2 uvrA were treated with the test material PRECAL 50S (Calcium hydroxide (hydrated lime)) diluted in 1M N-(2-Hydroxyethyl)piperazine-N-2-ethane sulfonic acid (HEPES) using the Ames plate incorporation method according to OECD Guideline No. 471. The substance was tested up to 3750 µg/plate with and without metabolic activation. No significant increases in the frequency of revertant colonies were recorded for any of the strains, with any dose of the test material, either with or without metabolic activation (Sokolowski, 2007).

An in-vitro chromosome aberration test was performed with calcium hydroxide in human dental pulp D824 cells. The occurrence of chromosome aberrations was investigated in the presence and absence of metabolic activation (rat liver S9-mix). For the main experiment test substance concentrations of 30, 100 and 300 µM were used. Calcium hydroxide did not induce a significant increase in the number of phases with aberrations at any preparation time and dose level. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed. In conclusion, Calcium hydroxide does not induce chromosome aberrations in D824 cells, neither in the presence nor in the absence of a metabolic activation system, under these experimental conditions (Nishimura et al., 2008).


Short description of key information:
Overall, the read-across from aluminium and calcium compounds within a weight of evidence approach does not support a systemic mutagenic hazard for “Reaction product of thermal process between 1000°C and 2000°C of mainly aluminium oxide and calcium oxide based raw materials with at least CaO+Al2O3+MgO >80% , in which aluminium oxide, magnesium oxide and calcium oxide in varying amounts are combined in various proportions into a multiphase crystalline matrix” and there are also no clastogenic effects in valid in-vivo studies.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the read-across from aluminium and calcium compounds, no classification is required according to DSD or CLP classification criteria for genotoxicity.