Sodium ethanolate

Endpoint summary

Additional information

No data/information is available for sodium ethanolate concerning genetic toxicity. But data are available for ethanol.

In vitro studies

Multiple studies exist with data on the potential of ethanol to cause reverse mutation in the commonly used bacterial strains (Ames test) both with and without metabolic activation (Zeiger et al., 1992; Blevins and Tylor, 1982; Blevins and Shelton, 1983; Phillips and Jenkinson, 2001; McCann et al., 1975; De Flora et al., 1984 a, b, c). Apart from one test which gave ambiguous test results in one strain (TA 102) and without cytotoxicity data, all other tests up to a plate concentration of 10 mg/plate showed no evidence of mutagenicity in the used 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 (Hayes et al., 1985) and testing was only carried out without metabolic activation. For that reason, effects were not regarded as adverse.

In a study published by Hellmer et al. (1982), DNA repair deficient and proficient strains of E coli bacteria were used in a DNA repair assay conducted 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.

Overall, it can be concluded with confidence that ethanol is not mutagenic to bacteria.

In several studies, the clastogenic potential of ethanol was tested in the following cell lines: human peripheral lymphocytes and Chinese hamster lung (Banduhn et al., 1985; Lin et al., 1989; Phillips et al., 2001)(). Ethanol was used at doses ranging from 1 - 5 %. No evidence for a clastogenic potential could be found with and without metabolic activation.

In a study published by Kayani et al. (2010), the recently developed CBMN (Cytokinesis Blocked Micronucleus) Assay was used. Ethanol in the absence of metabolic activation caused a statistically significant increase in numerical chromosome alterations at concentrations above 3 mg/ml (or 0.067M).

In three cell mutation studies using mouse lymphoma L5178Y cells (Phillips et al., 2001; Amacher et al., 1980; Wagenheim et al., 1988), 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. In a mammalian cell mutation study using S49 mouse lymphoma cells, ethanol was found to be non-mutagenic with activation at a dose of 0.17 M (Friedrich and Nass, 1983). It can be concluded that there is little evidence for the clastogenicity of ethanol in vitro.

In vivo studies

Micronucleus test

In a study published by Balansky et al. (1993), male rats were exposed to 5% v/v or 10% ethanol in drinking water (equivalent doses: 2.0 and 3.9 g/kg) for 10 days, 23 days or 30 days. No changes in the frequency of micronucleated polychromatic erythrocytes and pulmonary alveolar macrophages were found. However, ethanol significantly enhanced the number of binucleated and polynucleated pulmonary alveolar macrophages, possibly reflecting some generic disturbances in cell cycle control and nuclear-cytoplasmic balance although this is not clearly defined.

Some positive results have been reported for this end point (Baraona et al., 1981; Cebral et al., 2011).  Rats were fed for 6 weeks a liquid diet containing 36 % of total energy as ethanol (blood concentration ~150 mg/dl). Treatment significantly increased the frequency of micronuclei in bone marrow erythrocytes and the effect was associated with reduced number of nucleated cells and erythrocyte macrocytosis. In contrast, acute administration of ethanol did not produce any changes in bone marrow. A mouse micronucleus drinking water study in mice examined the impact of prolonged exposure to a single dose of more than 20 g/kg/day ethanol. A positive result was obtained but only at this single very high dose over an extend period of time not considered relevant to occupational exposure conditions.

In a study described by Chaubey et al. (1977), male mice were exposed to ethanol in drinking water at concentrations of 10 % or 20 % for 6 days, which was gradually increased to 20 % or 30 %, respectively, for the next 7 days, and finally to 30 % or 40 %, respectively, for the last 14 days of the study (estimated doses: 36 and 48 g/kg, respectively). Ethanol treatment did not increase the incidence of micronuclei in polychromatic and normochromatic. The doses used in this study were above normal recommended guideline maxima.

Chaubey et al. (1977) reported an in vivo micronucleus test using male mice treated with ethanol (CAS: 64-17-5) in the drinking water. Concentrations were up to about 65 g/kg bw/day; study duration was 27 days. A negative result was found.

Chromosome aberration

Data are available for Chinese hamsters published by Korte et al. (1981 a). Male and female Chinese hamsters received ethanol in drinking water at 10% v/v during the first week, 15% v/v during the 2nd and 3rd, and 20% v/v for additional 8 weeks (equivalent to 31g/kg for males and 37g/kg for females). Analysis of bone marrow cells did not show any increase in chromosomal aberrations and sister chromatic exchanges in the ethanol.

In another study reported by Korte et al. (1981 b), animals were exposed to 10 %v/v ethanol as their only liquid supply for 46 weeks (equivalent to ca. 10 g/kg/day). No treatment-related effects were observed in the frequency of chromosomal aberrations in peripheral lymphocytes, or in the number of SCE per metaphase in bone marrow cells.

In a study reported by Korte et al. (1981 c), male and female Chinese hamsters were exposed to 10 %v/v ethanol in drinking water for 9 weeks (equivalent to around 10 g/kg/day ethanol). Ethanol showed no effects on bone marrow chromosomes.

Dominant Lethal Assay

In an interlaboratory study of the dominant lethal assay, three laboratories performed studies using the same guideline protocol (James and Smith, 1982). Here, male mice received 0.16 g/kg/day (MTD) or 0.63 g/kg/day (MTD x 0.25) by gavage for 5 consecutive days and were paired with untreated females. There were occasional increases in the number of postimplantation deaths per male which mostly failed to be significant. Administration of cyclophosphamide, a known dominant lethal mutagen, consistently produced a significant increase in post-implantation death. The authors of the study concluded that ethanol is unlikely to produce a dominant lethal effect up to the maximum tolerated dose. In a study described by Randall et al. (1982), male mice were pair-fed isocaloric diets containing 0%, 10% or 20% ethanol for 4 weeks and then paired with untreated females. Applied doses were 14 -17 and 24 -31g/kg of ethanol, respectively. No differences were found between the litters of alcohol-treated males and controls in terms of number of implantation sites, prenatal mortality, foetal weight, sex ratio or frequency of soft tissue malformations.  Rao et al. (1994) reported a study, where three strains of male mice (Swiss, CBA and C57BL6) received 0.1 ml of 40% ethanol intraperitoneally once a day for three consecutive days (dose ca. 1.05 g/kg) and afterwards were paired with Swiss females. No dominant lethal effects were observed in two of the strains under acute ethanol treatment and the strain subject to sub-chronic treatment (C57BL6). However, the number of pregnancies and live and total implants were decreased in the ethanol-treated mice and the mutagenic index was consistently positive across all matings.

Mankes et al. (1982) published a study where male rats were administered ethanol as 20 % v/v in drinking water for 60 days (equivalent to 7.9 g/kg). Treatment resulted in a decrease in testicular weight and pathological changes in the testis and litter size and weight were reduced. Total embryonic deaths were increased and litter of ethanol treated group showed a higher incidence of soft-tissue abnormalities.

Chauhan et al. (1980) reported a study where the dominant lethal assay was performed in rats. Two groups of males received 15 % v/v ethanol in drinking water for 5 days which was increased gradually to 20 % v/v and 30 % v/v, respectively, for a period of 35 days. A third group was fed 30 % v/v ethanol (equivalent to 12 g/kg/day) for 4 days. Following treatment the animals were paired with females. There were no significant differences in the number of dead, live and total implantations at the prior or post-implantation.

In a study reported by Berryman et al. (1992), adult male mice were treated with ethanol (5 %v/v) as part of a liquid diet (28 % ethanol-derived calories) for 5 weeks followed by mating with females. The treatment resulted in a small decrease in testicular weight but minimal impairment of fertility. Ethanol treatment increased the incidence of dominant lethal mutations. The equivalent dose was 16 -22 g/kg ethanol. Such doses are well in excess of those relevant to occupation or consumer use of ethanol.

In contrast, in another study (Klassen et al., 1976), male rats received 6 % v/v ethanol containing liquid diet (providing 35 % of calories) which was increased to 10 % after 1 week (58 % of dietary calories -equivalent to 9 g/kg). Here, positive results could be shown. Ethanol treated animals showed signs of intoxication and weight loss compared to controls. Treatment with ethanol reduced the number of successful matings, litter number, and increased the incidence of early resorptions.

In the Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster three commercial mouthwash products containing 6%, 9% and 16.8% ethanol were tested. Ethanol produced significant increases in total spot frequency in flies with normal bioactivation capacity exposed to 12.5% (and above), and in flies with increased biotransformation capacity exposed to 8.4% (and above). Mouthwash products containing 6 and 9% ethanol failed to show genotoxicity in this assay. Results from marker-heterozygous and balancer-heterozygous flies exposed to a mouthwash product containing 16.8% ethanol suggested that the genotoxicity was due to mitotic recombination (Rodrigues et al., 2007).

The majority of the available studies are limited due to for example inadequate numbers of animals or the methods used to score or evaluate the incidence of early or late fetal deaths (in most cases, no distinction is made between early and late deaths). In addition, in most cases, interpretation is also compromised by the general toxic effects that were induced by the very high applied doses, which are well in excess of guideline recommendations, occupational and consumer use.

For the assessment of sodium ethaolate, it has to be considered that due to the caustic properties, secondary exposure to ethanol is considered to be low so that toxicity from ethanol will not occur.

Short description of key information:
Bacterial reversion mutation (Ames, S tymphimurium, strains TA97, TA98, TA100, TA104, TA1535, TA1537, TA1538): 7 negative assays, one of them includes multiple studies.
Bacterial reversion mutation (E Coli, non standard assays): one positive at cytotoxic concentrations, one negative.
Cytogenetic: 3 negative studies, not all stuies were performed with metabolic activation.
Mammalian cell gene mutation: 4 negative studies, with and without metabolic activation (not all studies use all combinations).

Endpoint Conclusion:

Justification for classification or non-classification

Due to the overall results obtained in in vitro- and in vivo-studies from ethanol, no classification is proposed.