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Disregarded studies:

A large number of in vitro and in vivo genotoxicity studies is available for anhydrous and basic aluminium chloride as well as for aluminium chloride hexahydrate; however, many of these show major deficiencies with regard to study design or experimental details and were disregarded after careful reviewing. These studies were, nonetheless, included in the dossier and are listed in the following paragraphs:

JETOC (1996) reported a bacterial reverse mutation assay using S. typhimurium strains TA 98, 100, 1535, 1537, 1538 and the E. coli WP2 uvrA strain. Test concentrations used were between 20 - 5000 ug/plate. A positive result was reported for two out of six bacterial strains (S. typhimurium TA 100 and 98) with and without metabolic activation. However, the reliability of the study is considered to be low since the results were not reported in the form of a comprehensive study and only two replicates per concentration were tested, which significantly decreases the statistical power of the assay. Furthermore, only single plates were used for the vehicle control group, no positive control data and no historical control data were reported, and finally, the positive response was very and was not confirmed in a second experiment. Therefore, the study was disregarded.

Lima et al. (2007) reported an in vitro mammalian chromosome aberration assay. Cultured human lymphocytes were treated with 5, 10, 15 and 25 uM aluminum chloride during the G1, G1/S, S (pulses of 1 and 6 h), and G2 phases of the cell cycle. No data are available about metabolic activation. All tested concentrations were cytotoxic, far beyond the range recommended in OECD guideline 473, and reduced significantly the mitotic index in all phases of cell cycle. The results are therefore considered unsuitable to assess a chromosome aberration potential of the test compound, and the study was assigned a Klimisch rating of 3 and disregarded.

Lankoff et al. (2006) conducted an in vitro comet assay to analyze the level of DNA damage in human peripheral blood lymphocytes treated with aluminium chloride hexahydrate (CAS 7784-13-6) and the impact of Al on the repair of DNA damage induced by ionizing radiation. Cells were treated with different doses of aluminium chloride (1, 2, 5, 10, and 25 µg/mL) for 72 h. Results indicated that Al induces DNA damage in a dose-dependent manner, however, at the dose of 25 µg/mL the level of damage declined. This decline was accompanied by a high level of apoptosis indicating selective elimination of damaged cells. Under the test conditions, aluminum chloride was considered to induce DNA damage in human peripheral blood lymphocytes by the authors. However, this study was conducted in non-GLP conditions and without standard methodology. Cytotoxicity was indirectly measured by apoptosis. The increase in DNA damage and apoptotic cells (cytotoxicity) was well correlated which means that the DNA damage seen could simply be a side-effect of cytotoxicity. Moreover, cells were exposed continuously for 72 h, therefore only toxicity-induced cell-cycle delay may have been observed. Also, no positive control was used. Therefore, the study was disregarded.

Turkez et al. (2010) performed an in vivo micronucleus assay with aluminium chloride hexahydrate. Rats were orally administered the test substance at 34 mg/kg bw/day for 30 days. A control group and 2 additional groups were also included: propolis at 50 mg/kg bw/day, and aluminium chloride (34 mg/kg bw/day) plus propolis (50 mg/kg bw/day). At the end of the experiment, rats were sacrificed and hepatocytes were isolated for counting the number of micronucleated hepatocytes. In addition, the levels of serum enzymes and histological alterations in liver were investigated.

Aluminium chloride induced a statistically significant increase in the numbers of micronucleated hepatocytes whereas propolis did not. Simultaneous administration of propolis attenuated the increased numbers of micronucleated hepatocytes induced by aluminium chloride. A significant increase in ALP, AST, ALT and LDH and induced histopathological changes in the liver were observed in the group treated with aluminium chloride.On the contrary, treatment with propolis alone did not cause any adverse effect on above parameters. Moreover, simultaneous treatment with propolis significantly modulated the toxic effects of aluminium chloride.

Under the test conditions, repeated oral administration of aluminium chloride was reported to induce a statistically significant increase in the number of micronucleated hepatocytes. However, this study was diregarded because of the following main limitations: Significant damage in the liver was reported (increase in levels of serum enzymes and histological alterations in liver) whereas in a GLP OECD guideline 422 study on AlCl basic (CAS 1327-41-9), no liver damage and reduced ALP activity were observed up to 1000 mg/kg/day. This discrepancy undermines the results found in this study. Also, the liver cannot be used as a target tissue for the micronucleus assay in adult rats: in this study, 8-weeks old adult rats have been treated for 30 days which means they were 12-weeks old at termination. This is too old for an appropriate micronucleus test in liver because hepatocytes divide too slowly at this age.

In a study by Geyikoglu et al. (2012), rats were intraperitoneally administered basic aluminium chloride at 5 mg/kg bw/day for 10 weeks. Control group was treated with sodium chloride under similar test conditions. At the end of the experiment, rats were sacrificed and hepatocytes (HEP) were isolated for counting the number of micronucleated hepatocytes (MNHEPs). In addition, haematological, biochemical parameters and histological alterations in liver and kidney were investigated. In the study, aluminium chloride induced a statistically significant increase in the numbers of micronucleated hepatocytes. In addition,the enzymatic activities of ALP, AST, ALT and LDH, and the levels of urea and uric acid significantly increased. RBC, WBC, PLT, Hb and Ht revealed significant decreases in the Al treated group compared to the control. Furthermore, severe pathological damages were established in both liver and kidneys of Al treated rats. The study basically showed the same limitations as the Turkez study and was disregarded for the same reasons.

Manna et al. (1972) performed an in vivo bone marrow chromosomal aberration test, mice were applied basic aluminium chloride intraperitoneally at dose-levels of 0.01, 0.05 and 0.1 M. Bone marrow cells of specimens at 0.1 M were fixed at 1, 2, 4, 8, 12, 16, 20, 24, 48 and 72 h after the injection while those of 0.01 and 0.05 M series were fixed at 20 h only after the injection. Four mice were used for each fixation interval. Specimens injected with distilled water and their bone marrow fixed at the corresponding intervals served as controls. Sodium citrate-acetic acid alcohol - air drying - Giemsa staining methodology was followed for the preparations of bone marrow cells.  The data of the treated series showed that the chromatid type breaks, gaps and constrictions were regular in occurrence while the chromosome type break was very rare. In the 0.1 M series, the aberration types would not show regularity in their increase or decrease at different fixation intervals. The data of 8 and 48 h were disregarded because of the inadequate figures; the frequency of total aberration remained more or less the same at different intervals between 4 and 72 h. Tissue fixed at one hour after the treatment had also a good number of chromosomal aberrations. The effect of AlCl3 on bone marrow chromosomes of mice was, therefore, non-delayed type and it continued without much change even at 72 h. In the combined data of 0.1 M series among the different types of aberration, the frequency per cell was found to be the highest for the chromatid type (0.1) and lowest for the chromosome type break (0.0024). The frequency of gaps and constrictions (0.0775) was close to that of the chromatid type. Treatment of 0.01, 0.05 and 0.1 M solutions of AlCl3 and the tissues fixed at 20 h revealed that the frequencies of total aberrations were 12, 20 and 26 %; the chromatid type breaks were 4.5 and 13.5 % and the gaps and constriction were 4, 6.7 and 9.5 %, respectively. Thus the values of the aberration frequency were not proportional to the different molar solutions used. However, the use of higher concentration of AlCl3, no doubt, increased the aberration frequency to some extent.   Under the test conditions, AlCl3 was considered to induce chromosomal aberrations at higher concentrations. However, this study suffers from many limitations: the early sampling times (just several hours after exposure) are unappropriate: it is biologically unrelevant to search for chromosome aberrations only few hours after administration. Also, the OECD guideline 475 recommends the analysis of at least 100 cells/animal : in this study, there were supposed to be 4 mice per group and 200 cells per group, therefore potentially only 50 cells analysed per animal, but in some cases even 200 cells could not be analysed, which suggests a serious problem with the quality and number of metaphases studied. Moreover, the volume administered intraperitoneally was 3.3 mL/100 g bodyweight which clearly exceeds the recommended limit volume of 2 mL/100 g bodyweight. In addition, there was no time-related change in aberration frequency which undermines the isolated increases observed. On top of that, cytotoxicity was not assessed and no positive control was used. The study was therefore disregarded.

A micronucleus test in Swiss mice was performed by Paz et al, 2017, in which aluminium chloride was administered to male and female Swiss mice orally at doses of 49, 98 and 161 mg/kg bw. Negative (distilled water) and positive (CPA) control groups were included as well. 24 h after administration, the animals were killed, bone marrow slides were prepared and evaluated for micronucleus frequency. Histopathological analysis was performed on livers, kidneys and stomachs, and the weight of these organs was determined.

The authors report a significant increase in the number of MN in all treated groups. Furthermore, the authors state that animals in all treated groups were "found to have histopathological alterations in the tissue integrity of their stomachs, kidneys and most of all their livers".

The study has several severe deficiencies making it unreliable:

Positive control results were originally not reported and only delivered upon request, and no historical control data are reported. The rationale for selecting the applied dose levels (fractions of a published LD50) is not in line with the OECD TG, which requires testing of a defined MTD. Clinical signs or body weights were not reported, which makes an evaluation of whether the MTD has been met or exceeded impossible. The test substance identity is unclear (indicated as "aluminium chloride hexahydrate" with CAS no 7446-70-0, but that CAS no. identifies anhydrous aluminium chloride and not the hexahydrate).

Most importantly, the reported histopathological findings are implausible: The animals were killed and their organs prepared for further examinations 24 hours after test substance administration. It is highly unlikely that the observed histopathological changes would manifest themselves within the extremely short period of 24 hours. Histopathology was not performed or not reported for the negative control group, so it is not possible to compare the treated and untreated groups. All in all, it is very likely that the observed effects are not related to test substance exposure but were present in all animals (from treated and control groups) already before substance administration. Furthermore, the microscopic alterations are not in line with the results of available high-quality studies, such as the combined 28-day repeated dose toxicity study and reproduction/developmental toxicity screening test of aluminium chlorid basic in rats (NOTOX, 2007; see IUCLID section 7.5): None of the histopathological changes observed by Paz et al. were seen in the NOTOX study from 2007, despite the fact the animals in this study were repeatedly treated with much higher aluminium doses for a much longer time.

All in all, the study is considered unreliable for the above-mentioned reasons and is disregarded.

Valid studies:

To assess the genotoxic potential of aluminium chloride, several valid in vitro and two valid in vivo genotoxicity studies, partly conducted with other aluminium salts as supporting substances, were evaluated in a weight-of-evidence approach.

The studies cited in IUCLID chapter 7.1.1 (ToxTest 2010; Priest 2010) demonstrate very similar systemic bioavailabilities of a number of aluminium compounds, including aluminium chloride and aluminium hydroxide. It has been postulated that especially those aluminium compounds that are water soluble will behave very similarly regarding bioavailability. Consequently, ECHA has agreed that "a joint assessment of AC, ACH and AS is justified based on read-across" (SEV-D-2114385103 -55 -01/F) and has performed substance evaluation of these three water-soluble aluminium salts jointly.

Therefore, read-across from aluminium hydroxide, basic aluminium chloride and dialuminium chloride pentahydroxide (which is also a soluble aluminium salt) was considered appropriate to cover the endpoint of genotoxicity for anhydrous aluminium chloride in accordance with section 1.5 in REACH Annex XI.

A valid Ames assay conducted in accordance with GLP and OECD guideline 471 was performed by BASF (BASF, 2015) and gave no indication of mutagenicity of anhydrous aluminium chlorid in S. typhimurium TA 98, 100, 1535, 1537, 1538 and the E. coli WP2 uvrA strain with and without metabolic activation.

Oberly et al. (1982) reported limited information from an in vitro mammalian cell gene mutation assay. Mouse lymphoma L5178Y cells were used. The test concentrations were 570, 580, 590, 600, 620 and 625 ug/ml. Anhydrous aluminium chloride was found to be non-mutagenic with and without metabolic activation.

Basic aluminium chloride was tested in a GLP-compliant in vitro micronucleus assay conducted according to OECD guideline 487 (NOTOX, 2010). The results of the study gave no indication of a clastogenic or aneugenic potential of the test substance.

Basic aluminium chloride was also tested in a GLP-compliant in vitro mammalian gene mutation assay (mouse lymphoma assay) conducted according to OECD guideline 476 (NOTOX, 2010) . A mutagenic potential was not identified in this study.

Villarini et al. (2017) assessed the early effects of co-exposure to 50 Hz ELF-MF (extremely low frequency magnetic fields) and non-cytotoxic doses of Aluminum chloride (AlCl3) on DNA damage by comet assay in SH-SY5Y and SK-N-BE-2 human neuroblastoma (NB) cells. Although the study was not specified as GLP-compliant, it is well documented and was performed according to a peer reviewed protocol developed during the International Workshop on Genotoxicity Test Procedures. The study gave no indication of DNA damage by aluminium chloride.

Aluminium hydroxide, a compound related to aluminium chloride, was tested in a GLP-compliant in vivo micronucleus assay conducted according to OECD guideline 474 (Covance, 2010). The test substance did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 2000 mg/kg/day (the maximum recommended dose for this study).

The potential of dialuminium chloride pentahydroxide to cause micronuclei in vivo was evaluated in a GLP-compliant in vivo micronucleus assay conducted according to OECD TG 474 (Stammberger, 1999) in mice treated up to 2000 mg/kg/day (the maximum recommended dose for this study).

The substance did not cause an increase in the the number of polychromatic erythrocytes containing micronuclei. Systemic bioavailability of the administered test substance was demonstrated by clinical signs (reduced spontaneous activity).

Summary:

None of the available studies considered valid and reliable gave an indication of a genotoxic potential of anhydrous aluminium chloride.


Justification for classification or non-classification

By means of the data available concerning genetic toxicity, a classification of the test substance is not warranted.