Magnesium ethanolate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Magnesium ethanolate rapidly hydrolyzes in aqueous environments (t1/2< 1 minute) into ethanol and magnesium hydroxide (detailed description in section 5.1.2). Valid in vitro study data on genetic toxicity are available for the hydrolysis products magnesium hydroxide and ethanol.

 

Hydrolysis product magnesium hydroxide

Three key studies were carried out on magnesium hydroxide: gene mutation in bacteria, cytogenicity in mammalian cells and gene mutation in mammalian cells. The results of all these studies were negative.

 

Hydrolysis product ethanol

Bacterial reverse mutation.

Multiple studies exist with useful data on the potential of ethanol to cause reverse mutation in the commonly used bacterial strains (Ames test) both with and without metabolic activation. Testing is frequently done in association with the use of ethanol as an ‘inert’ vehicle in the testing of other chemicals. Test results are frequently available for tests up to a plate concentration of 10 mg/plate. There is no evidence of mutagenicity in the strains TA97, TA98, TA100, TA104, TA1535, TA1537, TA1538).

 

In a non-standard bacterial forward mutation assay using E coli, ethanol was found to be genotoxic but only at the same concentrations of 15 -20% by volume that were also associated with profound cytotoxicity. Testing was only carried out without metabolic activation. Bearing in mind the massive concentration at which this result was seen and the conjunction with cytotoxicity leads to the conclusion that genotoxicity is not an important characteristic of ethanol. In an assessment of the potential mutagenic potential of ethanol, DNA proficient and deficient strains of E coli bacteria were used in a DNA repair assay. A liquid micromethod procedure to determine the minimum inhibitory concentration was used along with two other techniques (a spot test and treat-and-plate method). Test were carried out both with and without metabolic activation. The liquid micromethod (with and without metabolic activation and the spot test both gave negative results as did the treat-and-plate method without metabolic activation. The reported result with the latter with metabolic activation was equivocal, but there was insufficient information available to judge the significance of this.

 

Of particular interest as a summary of the genotoxicity of ethanol, the results from using ethanol as a vehicle in a number of guideline reverse bacterial mutation (Ames) assays was reported. Ethanol as a control vehicle, was used in the Salmonellas/microsome test at 200 ul/plate in the plate incorporation assay and up to 100 ul/plate in the pre-incubation assay, the latter being equivalent to 79 mg/plate, or approximately 16 times the normal maximum recommended dose level of 5 mg/plate used in regulatory mutagenicity tests. In 1998 it was used as the vehicle for 18 test materials. The mean, minimum and maximum frequencies of revertant colonies for the ethanol vehicle control plates were all comparable to the 1998 vehicle control history profile (320 -340 assays) for all vehicle controls used at this testing laboratory. Overall, it can be concluded with confidence that ethanol is not mutagenic to bacteria.

 

Clastogenicity

The clastogenic potential of ethanol has been examined in numerous assays and using a number of different mammalian cell lines, several of which are reported in this dossier. Concentrations tested are usually very high (eg 1-5%), well in excess of those normally recommended, and results are generally negative. Where positive results are reported, these are close to or exceed the normal maximum recommended concentrations for guideline studies. Many studies, typical of most, are usually incomplete because metabolic activation is not used.

 

In a review of the genotoxicity of ethanol, the results from using ethanol as a vehicle in a number of guideline clastogenicity (chromosome aberration) in vivo assays were reported for two cell lines (human lymphocytes and Chinese hamster lung). As a control vehicle, ethanol was used in the guideline tests at doses of 1%, well in excess of the maximum dose normally recommended for use. In 1998 across the two cell lines, it was used as the vehicle for 19 test materials without metabolic activation and 11 with. The mean, minimum and maximum frequencies of revertant colonies for the ethanol vehicle control plates were all comparable to the 1997 vehicle control history profile (145 assays) for all vehicle controls used at this laboratory during this year.

 

From the evidence, it can be concluded that there is little evidence for the clastogenicity of ethanol in vitro.

 

Mammalian cell mutation

Two similar cell mutation studies using mouse lymphoma cells in the TK forward mutation assay reported similar results; ethanol was found to be non-mutagenic with and without metabolic activation at very high doses up to and including those that cause significant cytotoxicity (typically in the region 0.3 -0.6M. In a mammalian cell mutation study using S49 mouse lymphoma lymphoma cells that monitored simultaneously selection for ouabain, 6-TG and dexamethasone resistance, ethanol was found to be non-mutagenic with activation at a dose of 0.17 M.

 

In a review of the genotoxicity of ethanol, the results from using ethanol as a vehicle in an in vitro guideline mammalian gene mutation assay was reported. As a control vehicle, ethanol was used in the guideline tests at a dose of 1%, well in excess of the maximum dose normally recommended for use. The mean, minimum and maximum mutation frequencies for the ethanol vehicle control plates were all comparable to the pooled results from 20 previous vehicle controls used.

 

The results from guideline gene mutation assays with ethanol are universally negative. In a study using the more recently developed CBMN (Cytokinesis Blocked Micronucleus) Assay, ethanol in the absence of metabolic activation, was found to cause a statistically significant increase in numerical chromosome alterations (aneuploidy) at concentrations above 3mg/ml or 0.067M). In contrast, when the metabolite acetaldehyde was tested, it causes chromosomal alterations (clastogenicity) at concentrations of < 0.04 mg/ml. It should be noted that the maximum recommended dose for testing chemicals in current standard in vitro protocols is 5 mg/ml or 0.01 M, which this study clearly exceeded in a number of the tested doses of ethanol.

Justification for classification or non-classification

Magnesium hydroxide tested negative in 3 key in vitro studies.

There is no significant evidence that ethanol is a genotoxic hazard according to the criteria applied normally applied for the purposes of classification and labelling, when data that is only applicable to heavy consumption of alcoholic beverages is excluded, the limit doses normally applied in guideline studies are taken into consideration and the fact that confounding due to other toxic effects associated with very high doses are accepted.

Magnesium ethanolate therefore does not meet the criteria for classification.