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IN VITRO

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 10mg/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 Salmonells/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 a number of assays and using a number of different mammalian cell lines, a number 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 abberation) 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 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 0.17M.

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 - yet to become guideline, 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 cause chromosomal alterations (clastogenicity) at concentrations of <0.04mg/ml. It should be noted that the maximum recommended dose for testing chemicals in current standard in vitro protocols is 5mg/ml or 0.01M, which this study clearly exceeded in a number of the tested doses of ethanol.

IN VIVO

Micronucleus test

Male mice were exposed to ethanol in drinking water at concentrations of 10% or 20% for a period of 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. Weighted time averaged doses were estimated to be 36 and 48g/kg respectively. Ethanol treatment did not increase the incidence of micronuclei in polychromatic and normochromatic erythrocytes in contrast to the positive control, ethylmethanesulfonate, which induced a significant increase in polychromatic cells.  The doses used in this study were well above normal recommended guideline maxima.

Male rats were exposed to 5% v/v or 10% ethanol in drinking water for a period of 10 days, 23 days or 30 days in three separate experiments. These were equivalent to doses of 2.0 and 3.9g/kg. The treatment did not induce any changes in the frequency of micronucleated polychromatic erythrocytes and pulmonary alveolar macrophages. 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. A dose of 5% for 10 days is within the guideline but the other doses were about 2x those normally recommended (on a dose/time basis).

Some positive results have been reported for this end point. Rats were fed for 6 weeks on a liquid diet containing 36% of total energy as ethanol designedto produce blood ethanol levels similar to those observed in alcoholics. The treatment, which resulted in blood ethanol concentrations of ~150mg/dl, marginally but significantly increased the frequency of micronuclei in bone marrow erythrocytes compared with pair fed controls 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 very high dose of ethanol (>20g/kg/day). A positive result was obtained but only at a single, very high dose over an extend period of time not considered relevant to occupational exposure conditions. It should be noted that the levels of ethanol used in these studies were well in excess of those relevant to occupation or consumer use of ethanol and the results need to be put into this context.

Overall, there is no convincing evidence that ethanol induces micronuclei in the bone marrow of rodents.

Chromosome aberration

Reliable results are only available for Chinese hamsters.  Male and female Chinese hamsters were administered 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. Dosing was equivalent for the latter two thirds of the study of 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 group compared with the vehicle control group and at doses well in excess of what is normally recommended in the guideline.  In another study, animals were exposed to 10%v/v ethanol as their only liquid supply for 46 weeks (equivalent to ca. 10g/kg/day). At the end of the treatment no 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 third study, male and female Chinese hamsters were exposed to 10%v/v ethanol in drinking water for 9 weeks (equivalent to around 10g/kg/day ethanol).  Ethanol had no effect on bone marrow chromosomes in either sex.

Dominant Lethal Assay

In a collaborative a cross laboratory study of the dominant lethal assay conducted by the British Pharmaceutical Industry, three laboratories conducted studies using the same guideline protocol. Male mice received 0.16g/kg/day (MTD) or 0.63g/kg/day (MTD x0.25) by gavage for 5 consecutive days. After completion of the schedule the animals were paired with untreated virgin females, each week, for 8 consecutive weeks. All females were killed and examined 18 days after first being paired with males. Although there were occasional increases in the number of post implantation deaths per male, the majority of post implantation results were not significant and it confirmed that ethanol is unlikely to produce a dominant lethal effect up to the maximum tolerated dose. On the other hand, administration of cyclophosphamide, a known dominant lethal mutagen, consistently produced a significant increase in post-implantation death in the first two weeks of mating. The authors of the study concluded that ethanol is unlikely to produce a dominant lethal effect up to the maximum tolerated dose.  This study is considered the most relevant for this end point as it was to guideline and also tested using a dosing regimen that was both relevant to use of ethanol as a chemical intermediate or solvent and within the levels recommended by the guideline.  This study also established an MTD for this end point of less than 1g/kg in mice.

All other DLA studies available use doses well in excess of those recommended in the guidelines and gave both positive and negative results.

Male mice were pair-fed isocaloric diets containing 0%, 10% or 20% ethanol for 4 weeks and then paired with untreated females. The ethanol dosed animals received 14 -17 and 24 -31g/kg of ethanol respectively. Pregnant animals were examined on day 19 of gestation. 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.  In another study, three strains of male mice (Swiss, CBA and C57BL6) were administered 0.1ml of 40% ethanol intraperitoneally once a day for three consecutive days (dose ca. 1.05g/kg). Following treatment the animals were paired with virgin Swiss females on a 4 -day mating schedule for three sequential matings.. No dominant lethal effects were observed in two of the strains of mice under acute ethanol treatment and the strain subject to sub-chronic treatment (C57BL6) also failed to show any dominant lethal effects.  However, the number of pregnancies and live and total implants were decreased in the ethanol-treated Swiss mice compared to control mice and the mutagenic index was consistently positive across all matings for the Swiss mice. Similarly, in another study where male rats were administered ethanol as 20% v/v in drinking water for 60 days (estimated to produce a dose of 7.9g/kg), the treatment induced a decrease in testicular weight and pathological changes in the testis of ethanol treated animals. Litter size and weight was reduced in ethanol-treated animals. Total embryonic deaths were increased by ethanol treatment, and the litter in the ethanol group presented a higher incidence of soft-tissue abnormalities.

In a dominant lethal assay in rats, two groups of males were given ethanol 15% v/v in drinking water for 5 days which was increased gradually to 20% v/v and 30% v/v, respectively, for a total period of 35 days. A third group was fed 30% v/v ethanol for 4 days only. Dosing at 30% gives an approximate dose of 12g/kg/day. Following treatment the animals were paired with virgin females at weekly intervals and 8 sequential pairings undertaken. Females were examined for uterine contents and corpora lutea 10 -11 days after their separation from the males. There were no significant differences in the number of dead, live and total implantations at the prior or post implantation levels in the ethanol and control groups.  In contrast, another study in which male rats were exposed to 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 9g/kg) showed positive results.  Ethanol treated animals showed signs of intoxication and weight loss compared to controls. The treatment with ethanol reduced the number of successful matings, litter number, and increased the incidence of early resorptions compared to controls.

The majority of the studies available can be criticized on the grounds of inadequate numbers of animals or on 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 the majority of cases, interpretation is also compromised by the effects on fertility and likely other general toxic effects at the very high doses used, which are well in excess of guideline recommendations, even more so when considering the extended periods of treatment used.  The most satisfactory and reliable study is that interlaboratory evaluation described in the first paragraph.  On the basis of this and the fact that negative results have also been obtained at large doses, it can be concluded that ethanol is negative in the dominant lethal assay when tested according to guidelines appropriate for the evaluation of ethanol as a chemical substance.

DNA damage

In an in vivo test for potential to cause DNA damage, cohorts of pregnant mice (GD7) were given single gavage doses of ethanol at 2, 4 and 8g/kgbw and examined 4, 8, 12 and 24 hours post exposure for tisssue analysis. DNA damage was detected using the comet assay and was see in lungs, brains and embryos but not livers in the mid and high dose animals but not the low dose animals. Recovery from all effects was seen in the mid dose animals by 24hours. Mid dose animals showed some short term CNS effects and marked and persistent adverse CNS effects were seen in the high dose animals. The no effect level was 2g/kg, which is the maximum recommended dose in the guideline for single dose experiments (Kido, 2006). This result was confirmed in a later study that was part of a validation study of the in vivo rat alkaline comet assay, the ability of ethanol to induce DNA damage in the liver and glandular stomach of male rats was examined. No adverse effects were seen up to the maximum tested dose of 2g/kgbw. Positive controls and other genotoxic substance examined as part of the study confirmed the assay as functional (Nakagawa, 2015).


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

DNA damage: NOAEL confirmed in two studies as 2000mg/kgbw

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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.