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Key Studies

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E.coli strain WP2 uvrA were exposed to test material at up to the maximum recommended dose level, both in the presence and absence of metabolic activation system (rat liver S9-mix) using the plate incorporation method in two independent experiments.Vehicle and positive control groups were also included in mutagenicity tests. The vehicle and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Toxicity was observed in tester strains TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation in two independently repeated experiments. Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coliWP2uvrA.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test material in the presence and absence of a metabolic activation system (1.8 % (v/v) S9-mix). Dose range finding test was performed to select the appropriate dose levels for the cytogenetic assays. In the first cytogenetic assay, test material was tested up to 1000 and 700 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence and presence of S9-fraction, respectively. In the second cytogenetic assay, test material was tested up to 1500 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Cytochalasine B (5 μg/mL) was added to the cells simultaneously with the test substance. The cells were then treated with a hypotonic solution, fixed, stained and examined for toxicity and micronuclei. Vehicle and positive controls were also included in the study. The number of mono- and binucleated cells with micronuclei found in the vehicle control cultures was within the laboratory historical control data range. The positive control chemicals produced a statistically significant increase in the number of cells with micronuclei, demonstrating the sensitivity of the test system. Test material precipitated in the culture medium at 700 µg/mL and above concentrations. Test material did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Under the test conditions, test material is not clastogenic or aneugenic in human lymphocytes in the absence and presence of S9-mix.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline No. 476 and in compliance with GLP, L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to test material with a range of concentrations. Vehicle and positive control groups were also included in each mutation test. In the dose range finding test, after 3h of treatment without S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 725 μg/mL compared to the suspension growth of the solvent control. With S9-mix, the relative suspension growth was 66 % at the highest test substance concentration of 725 μg/mL compared to the relative suspension growth of the solvent control. After 24 h of treatment without S9-mix, the relative suspension growth was 45 % at the test substance concentration of 363 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 725 μg/mL. Test material precipitated in the exposure medium at concentrations of 333 μg/mL and above. In the mutagenicity test, the relative total growth was reduced by 90 % at 666 μg/mL compared to the total growth of the solvent controls at 24 h treatment without S9-mix. Test material did not induce a significant increase in the mutation frequency at any dose level either with or without metabolic activation in two independently repeated experiments. In all tests the concurrent vehicle and positive control were within acceptable ranges. Under the test conditions, test material is not mutagenic at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells, in the absence and presence of S9-mix.

Read-Across and Weight of Evidence/Disregarded Studies

A GLP, Klimisch Grade 1 in vivo micronucleus study on aluminium hydroxide has been used as a read-across study in support of the three key in vitro studies. It is considered that a read-across from aluminium hydroxide to aluminium sulphate is justified and robust because the two salts have similar solubilities. The RA in vivo study provided a negative conclusion for in vivo genotoxicity, which is in agreement with the Key in vitro study results on aluminium sulphate.

A total of six in vitro studies and 1 in vivo study have been included as weight of evdence or disregarded studies. Some of these studies provided negative results that were in agreement with the conclusions of the Key and RA studies. However, some of the studies also provided contrary positive conclusions. But none of the studies reported as positive were GLP and thus were classified as Klimisch grade 3 or 4. In addition, these studies were poorly executed and inaequately reported. The dose levels used were not clearly justified and the time-points used were different from the guideline recommendations. An expert review revealed that none of these studies was considered to be sufficiently reliable to add any significant weight against the strength of the Key and RA studies.


Justification for selection of genetic toxicity endpoint
Key studies that meet the requirements of REACH Annex VII and VIII.

Short description of key information:
The three key studies are:
An OECD 471 GLP Salmonella/E.coli bacterial mutation assay, an OECD 476 GLP mammalian cell L5178Y mutation assay and an OECD 487 GLP human lymphocyte chromosome aberration (micronucleus) assay in mammalian cells.
All three are GLP, Klimisch grade 1 studies and conducted according to the relevant OECD test guidelines and meet the Annex VIII test requirements of REACH. All three studies provide clear negative (non genotoxic) results. The dossier includes other study summaries of studies published in the literature. These are of mostly poor quality and are non-GLP with many deviations from recoqnised test methods and good scientific practise. Some of these studies are reported by the authors as showing positive (genotoxic) results. However, none of the data are reliable and in a weight of evidence approach they carry little weight in comparison to the Key studies.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008 including ATP3.

Self classification:

The Key studies show no positive results for mutagenicity or genotoxicity. In a weight of evidence approach the supporting studies are considered not to change the conclusion taken from the key studies.

Based on the available data, the substance is :

- not classified for mutagenicity according to the Regulation (EC) No. 1272/2008.

- not classified for mutagenicity according to the Directive 67/548/EEC.