Registration Dossier

Reference

Endpoint:: two-generation reproductive toxicity
Remarks:: based on test type (migrated information)
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 1985
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: other: Well reported study following NTP Protocol.
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:: yes
Remarks:: P generation no organ weights, sperm parameters or oestrous cycle included; P animals slightly older than recommended at first exposure; low number of pregnant females
Qualifier:: according to guideline
Guideline:: other: NTP Protocol. Fertility assessment by continuous breeding
GLP compliance:: not specified
Limit test:: no
Species:: mouse
Strain:: CD-1
Sex:: male/female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Charles River Breeding laboratories
- Age at study initiation: (P) animals 6 weeks at receipt, 11 weeks at first exposure.
- Fasting period before study: no
- Housing: 4-5 per cage by sex. In pairs during breeding and thereafter individually for 21 days.
- Diet (e.g. ad libitum): Pelleted feed (NIH-07 open formula rodent chow) ad libitum
- Water (e.g. ad libitum): deionized/filtered ad libitum
- Acclimation period: 2 weeks prior to preliminary range-finding study

ENVIRONMENTAL CONDITIONS
- Temperature : approx 21 C
- Humidity (%):
- Air changes (per hr): 12-14
- Photoperiod (hrs dark / hrs light): 10/14
Route of administration:: oral: drinking water
Vehicle:: water
Details on exposure:: PREPARATION OF DOSING SOLUTIONS: Ethanol administered in deionized, filtered water.
Details on mating procedure:: - M/F ratio per cage: 1:1
- Proof of pregnancy: litters were proof of pregnancy.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Samples of ethanol formulation in drinking water, control drinking water and bulk chemical were sent to Midwest Research Institute (Kansas City, MO), prior to preliminary range finding study, and at weeks 1, 6, 12, and 18 of main study with Parental animals.
Duration of treatment / exposure:: Exposure period: 18 weeks
Premating exposure period (males): Parental 7 days; F1 74 days
Premating exposure period (females): Parental 7 days; F1 74 days
Frequency of treatment:: ad libitum
Details on study schedule:: Number of generation studies: 2
Remarks:: Doses / Concentrations:
5, 10 and 15% v/v in water
Basis:
nominal in water
No. of animals per sex per dose:: 20 for P generation, also 20 F1 animals at the high dose mated at 74 days old.
Control animals:: yes, concurrent no treatment
Details on study design:: - Dose selection rationale: 14 day dose range finding study conducted. High dose for the 13 week study chosen such that depression of weight gain <10%
Positive control:: N/A
Parental animals: Observations and examinations:: DETAILED CLINICAL OBSERVATIONS: No. Only twice daily cage side inspections.

BODY WEIGHT: Yes
- Time schedule for examinations: at end of week 1, 2, 5, 9, 13 and 18.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: at end of week 1, 2, 5, 9, 13 and 18.
Oestrous cyclicity (parental animals):: Gestation index, changes in lactation and changes in oestrus cycles were not studied.
Sperm parameters (parental animals):: Epididymal and vas sperm were evaluated for concentration, motility and morphology in F1 males only.
Litter observations:: Litters were not standardized.
Postmortem examinations (parental animals):: Not conducted
Postmortem examinations (offspring):: GROSS NECROPSY
- High dose F1 animals had liver, kidney/adrenal and male sex organs weighed at termination.
Statistics:: Fertility and mating indices: Cochran-Armitage test for dose related trend and Fisher's exact test for comparisons between groups.
Size and number of litters, proportion of live pups and sex ratio, pup body weight, necropsy weight and sperm characteristics: Kruskal-Wallis for overall differences among groups, Jonckheere's test for dose related trends and Wilcoxon's test for pairwise tests.
Litter and dam weight: Williams' test
Reproductive indices:: Fertility indices were 97, 100, 100 and 94% in the controls and 5%, 10%, 15% ethanol groups respectively.
The F1 offspring of the 15% ethanol pairs had fewer live pups per litter. Unadjusted F1 live pup weight was greater for females and combined sexes at 5% but not at the higher concentrations. Body weights were lower than control in the 15% ethanol treated F1 offspring at mating and on day 21. Fertility indices in F1 matings were 85% and 65% in the controls and 15% ethanol groups respectively. Their F2 offspring weighed less as ethanol treated pups than control pups (males, females or both sexes). Other reproductive performance indices e.g. gestation index, changes in lactation and changes in oestrous cycles were not studied.
Offspring viability indices:: Proportion of pups born alive
Clinical signs:: not examined
Body weight and weight changes:: no effects observed
Food consumption and compound intake (if feeding study):: no effects observed
Organ weight findings including organ / body weight ratios:: not examined
Histopathological findings: non-neoplastic:: not examined
Other effects:: not examined
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: not examined
Reproductive performance:: no effects observed
Dose descriptor:: NOAEL
Effect level:: 15 other: % in drinking water
Sex:: male/female
Basis for effect level:: other: No effects observed in parameters studied at all doses
Clinical signs:: not examined
Mortality / viability:: not specified
Body weight and weight changes:: effects observed, treatment-related
Sexual maturation:: effects observed, treatment-related
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Gross pathological findings:: not examined
Histopathological findings:: not examined
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: 10 other: % in drinking water
Sex:: male/female
Basis for effect level:: other: At the highest dose fewer pups per litter were observed and significant changes to sperm motility
Dose descriptor:: NOAEL
Generation:: F2
Effect level:: < 15 other: % in drinking water
Sex:: male/female
Basis for effect level:: other: Lower live pup weight observed at the 15% dose studied
Remarks on result:: not determinable
Remarks:: no NOAEL identified
Reproductive effects observed:: not specified
Conclusions:: Overall, ethanol in drinking water at concentrations up to 15% (equivalent to 20.7 g/kg/day) had no demonstrable effect on fertility in this two-generation study.
Executive summary:: A two-generation study investigated the effects of 5%, 10% and 15% ethanol in drinking water in reproduction and fertility. Male and female CD-1 mice were continuously treated for 1 week prior to mating and for a 14 week breeding period followed by a 21 day holding period when they were separated and housed individually. The F1 offspring of the 15% ethanol pairs had fewer live pups per litter but ethanol treatment had no effect on the proportion of breeding pairs producing at least 1 litter during the continuous breeding phase or the number of litters per pair. The F1 offspring from the 15% group had decreased bodyweight at weaning and mating, and a decreased weight of testis, epididymides and seminal vesicles which was no longer evident when these were adjusted for body weight. There was also a significantly decreased percentage motile sperm but no changes in sperm concentration, and percentage of abnormal sperm or tailless sperm. When reproductive performance of F1 control and 15% ethanol-treated breeding pairs was assessed at 74 days of age, there was no significant difference in mating and fertility between the groups. However, adjusted live pup weight for the ethanol group was significantly reduced compared to controls which was likely due to generalized maternal toxicity.

Dose descriptor:

NOAEL

13 800 mg/kg bw/day

Dose descriptor:

NOAEC

30 400 mg/m³

Animal data

All available studies on ethanol use extremely high doses. Some establish NOAELs and clearly are the most useful, whilst others do not establish NOAELs at such very high doses. Clearly, the most useful studies are those that are closer to guideline (in terms of observations and number of dose levels used) and those that establish true NOAELs at the lowest doses. Such studies are given greater weight in the overall interpretation of the data for this end point.

The most reliable study performed to the most appropriate protocol and the one given the greatest weight as well as the key study is a two-generation study investigated the effects of 5%, 10% and 15% ethanol in drinking water in reproduction and fertility. Male and female CD-1 mice were continuously treated for 1 week prior to mating and for a 14 week breeding period followed by a 21 day holding period when they were separated and housed individually. The F1 offspring of the 15% ethanol pairs had fewer live pups per litter but ethanol treatment had no effect on the proportion of breeding pairs producing at least 1 litter during the continuous breeding phase or the number of litters per pair. The F1 offspring from the 15% group had decreased bodyweight at weaning and mating, and a decreased weight of testis, epididymides and seminal vesicles which was no longer evident when these were adjusted for body weight. There was also a significantly decreased percentage motile sperm but no changes in sperm concentration, and percentage of abnormal sperm or tailless sperm. When reproductive performance of F1 control and 15% ethanol-treated breeding pairs was assessed at 74 days of age, there was no significant difference in mating and fertility between the groups. However, adjusted live pup weight for the ethanol group was significantly reduced compared to controls which was likely due to generalized maternal toxicity.

All other available studies are incomplete in comparison to guidelines (usually either single sex treatment or truncated exposures). However, in total, they provide useful additional information.

In a fertility study, male Sprague-Dawley rats were exposed 7 hours per day for six weeks to 10,000 or 16,000ppm ethanol by inhalation and then mated with untreated female rats. Pregnant females received the same experimental treatment from day 1 -19 of gestation and were allowed to deliver their offspring. Treatment with ethanol did not affect the weight gain of parental animals. Incidence of fertility did not differ from controls and no group differences were found for litter size, number of dead pups, or length of pregnancy. Offspring survival and weight gain was also not affected by ethanol treatment. Adult male rats were kept in a chamber providing 22, 23, 25, or 27 mg/l ethanol air concentrations continuously for a 4 to 5 weeks period. At the end of the exposure period the animals were sacrificed and plasma testosterone levels and sex organ weights for testis, seminal vesicles and prostate were determined. Blood ethanol levels of = 130mg/100ml were compatible with adequate body weight and not associated with diminished plasma testosterone levels or reduced sex organ weights, whereas blood ethanol levels >=180 mg/100ml were associated with inhibition of testosterone secretion only in those animals who failed to grow. No adverse fertility effects were seen at the maximum dose, although weight loss was seen from 25mg/l and above.

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), estimated as 7.2 -14.4g/kg/day. After two weeks they were paired with untreated females during the hours of darkness when they had no access to food or drink. The treatment with ethanol for males continued during the hours of light for 5 weeks. All pregnancies were terminated on day 20 of gestation. Ethanol treated animals showed signs of intoxication and weight loss compared to controls from the start of the study, suggesting a LOAEL of 6% for such effects. The treatment with ethanol reduced the number of successful matings, litter number, and increased the incidence of early resorptions compared to controls.

Male rats were administered 2.5 or 5g/kg ethanol daily by gavage for 3 weeks or 9 weeks. Animals in the 2.5 g/kg and control groups were pair fed to those in the 5 g/kg group. Males were bred once after 3 weeks of treatment and twice after 9 weeks of treatment. Females in the first two breedings were sacrificed on day 20 of gestation and their offspring examined. Females in the third breeding were allowed to deliver their litters. There were no apparent treatment effects on resorptions or litter size. Fecundity was reduced in females bred to the high dose ethanol-treated males when all breedings were pooled. The number of male fetuses was increased in the high dose ethanol treated group. There was a significant dose related increase in fetal weights at the week 3 and 9 breedings and a significant increase in placental weights at the 9 week breedings. However, there were no treatment related effects on newborns sired by ethanol treated males. The main finding and only finding consistent and repeated across both breedings was an increase in fetal weights of offspring sired by alcohol treated males. It is not clear if this is an adverse toxicological finding.

Male adult rats were given a liquid diet with 36% of the daily calories derived from ethanol (approximately 13.6 g/kg bw/day), an isocaloric control diet, or standard laboratory chow, for 55 days. Reproductive function was assessed in two separate studies, following either natural mating, or with artificially inseminated females. Ethanol treatment impaired sexual behaviour, and only 22% of these rats reached ejaculation (compared with 50 and 70% in isocaloric and lab-chow controls, respectively). Fertility of ethanol-treated animals was significantly reduced, mainly following the natural matings. Ethanol treatment at this dose also significantly reduced serum testosterone levels, daily sperm production, and epididymal sperm count, associated with an acceleration of the sperm transit time in the cauda epididymis, decrease in sperm motility and increase in the percentage of abnormal shaped sperm cells.

Male mice were exposed to a nutritionally balanced diet providing 10% or 25% of ethanol-derived calories and mated with untreated females for 4 hours a day sequentially for 7 weeks. No toxic responses were noted in treated males other than decreased bodyweight gain at 25% ethanol-derived calories in diet. Paternal treatment did not affect fertility during the period studied nor litter size, or weight at birth or at weanling. The 25% dose is equivalent to 21.5g/kg ethanol. Male mice were treated with a diet containing 5% or 6% ethanol for 10 weeks and 5 weeks, respectively. After treatment with ethanol was completed, animals were hemicastrated (right testis and accessory organs) and were then left in an ethanol-free diet for 10 weeks to determine the recovery of reproductive effects. The treatment with ethanol diet induced a decrease in testicular weight and in seminal vesicle/prostate weight which was reversible at the end of 10 week treatment abstinence. Significant increases in frequencies of germ cell desquemation and of inactive seminiferous tubules were observed and these remained elevated except for the inactive seminiferous tubule levels in the 5% group which returned to control levels. Ethanol treatment affected the quality of spermatogenesis, caudal epididymal sperm content, sperm motility and in vitro fertilization of mouse oocytes but the changes disappeared after following 10 weeks abstinence. Forward progression of sperm was reduced in both treatments but persisted in the 6% group. No NOAEL was established (5% ethanol diet, 166mg% BAL). However, for persistent effects the NOAEL would appear to be close to 5% ethanol diet, which is estimated to be ~14g/kg/day.

Twenty-day old female rats were administered 2.5% or 5% ethanol in a liquid diet for period of 50 -55 days during which their oestrous cycle was determined. At the end of the treatment ovaries, uteri and vaginae were excised for histological examination. Ovarian function was suppresed only in the animals that received 5% ethanol as manifested by absence of oestrous cycles, a delay in vaginal opening, the absence of several generations of corpora lutea, inhibition of growth of the uteri and vaginae, and a reduction of ovarian and uterine weights. A NOAEL was established of approximately 8g/kg/day. Twenty eight-day old female rats were fed a liquid diet providing 36% ethanol-derived calories (5% of a liquid feed) or a pair-fed isocaloric diet for 49 days. This dose is estimated to be in the range 5.4 -11.4g/kg/day. At the end of the exposure period the animals were sacrificed and the uterus, fallopian tubes, ovaries, cervix, vagina and liver were macro- and microscopically examined. Ethanol-treated animals experienced a reduced weight gain compared to controls whilst their livers were larger and of fatty appearance. Serum liver enzymes, alkaline phosphatase, glutamic oxalo-acetic-acid-transaminase, glutamic pyruvic transaminase and gamma glutamyl transpeptidase were significantly increased compared with controls. Ethanol treatment reduced the weight of the ovaries, uterus and fallopian tubes compared with pair-fed isocaloric control group. Histological examination revealed differences in the appearance of uterus, cervix and vagina between treated and untreated animals, and absence of developing follicles, corpus lutea and corpus hemorrhagica in the ovaries of the treated animals. Compared with isocaloric controls, plasma estradiol and progesterone were reduced in ethanol-treated animals, whereas significantly higher plasma estrone levels were observed. Female rats were fed 5% ethanol in a liquid diet for 16 weeks, or for 8 weeks followed by laboratory chow and water for another 8 weeks. (Doses equivalent to 14 -21g/day and estimated to produce average serum ethanol levels of around 250mg%). Pair-fed controls and ad libitum fed controls were also included in the study. Vaginal patency was significantly delayed in the ethanol groups and irregular and longer oestrous cycles were noted in the 16-week ethanol treated group. After 16 week treatment females were mated with untreated males and their ethanol exposure was stopped till they delivered their litter. No adverse effect on fertility, litter size or neonatal bodyweight was detected.

Human data

The only data available is from the consumption of alcoholic beverages and care is required in the extrapolation of the results from such studies to the hazards from exposure by other routes than deliberate oral consumption.

In a study in young Danish men aged 18 -28 years, alcohol consumption did not appear to significantly affect male reproductive parameters at consumption levels <20 units per week.

A relatively small cohort of women were followed over two menstrual cycles to assess the relation between acute alcohol consumption, reproductive hormones, and markers of menstrual cycle dysfunction including sporadic anovulation, irregular cycle length, luteal phase deficiency, long menses, and heavy blood loss. The authors reported relatively small changes in a number of hormone levels for every 14g ethanol consumed, the authors reported that the geometric mean total and free estradiol, total and free testosterone, and luteinizing hormone were higher by between 1.5 to 6.2%, after adjustment for a number of potential confounding factors. However, the confidence ranges of the absolute values of all of the hormones did not differ significantly. The reported changes are very small in magnitude and it would be useful to know more the natural variation in these hormone levels over the menstrual cycle to judge whether the findings could be random statistical findings within the natural background variation. Moderate alcohol intake does not appear to have adverse short-term effects on menstrual cycle function, including sporadic anovulatio (Schliep, 2015)

A large prospective cohort study in Japan found that heavy alcohol consumption d(>300g/week ethanol) during the second and third trimesters, but not during the first trimester, is associated with increased risk of preterm delivery. The risk increased four-fold risk compared with non-drinkers (multivariable-fully corrected OR 4.52; 95% CI 1.68–12.2). This was not seen with lower level consumption and, indeed, light alcohol consumption (1–149 gethanol/week) tended to be associated with a reduced risk of preterm delivery, which was of borderline statistical significance. From this study it can be concluded there is no risk of pre-term delivery from exposure to ethanol other than via deliberate and excessive consumption of beverages above safe guideline levels (Ikehara, 2019).

Short description of key information:
Mouse (fertility unless stated)
- Key and most reliable study: NOAEL 13.8g/kg (pups/litter, sperm effects in F1 generation)
- NOAEL: 21.5g/kg (F1 male, other effects)
- NOAEL (male)>6g/kg
- LOAEL (male) ~14g/kg/day
Rat (fertility unless stated)
- NOAEC: >16000ppm (male and female), >16000ppm (male only), >14000ppm (male only) 12000ppm (males, weight gain)
- LOAEL: 7.2-14.4g/kg (fertility, weight loss), 7.2g/kg (intoxication)
- NOAEL: (female) 8g/kg
- LOAEL female <5.4-11.4g/kg depending on method of estimation.
- NOAEL: (male)>5g/kg: (female, fecundity): 2.5g/kg NOEL F1 (based on weight increase of fetus): <2.5g/kg
- NOAEL: (male)<13.8g/kg
Based on Blood ethanol concentrations for rat and fertility end point:
- NOAEL(male)= 127, 248, 338 mg/100ml BAC
- LOAEL (male) = 163 mg/100ml BAC; 166 (close to NOAEL)
- Rat female parental NOAEL <14-21g/kg/day, NOAEL F1=14-21g/kg/day
- LOAEL (female) = 110, 249, 250 mg/100ml BAC
- NOAEL (female rat fertility) = 155+/-9 mg/100ml BAC
- NOAEL (female rat fertility) < 110+/-9 mg/100ml BAC
- NOAEL (female rat fertility) <249mg/100ml (oestrus cycle disturbance)

NOAEC (inhalation, rat) maternal toxicity 16000ppm, teratogenicity >20000ppm
NOAEL (oral diet or drinking water, mouse) maternal toxicity ~13.7, <12, 16 g/kg, <23.7g/kg; teratogenicity 13.7, <12, 16, >23.7g/kg respectively
NOAEL (oral, gavage, mouse): maternal toxicity 2.2g/kg, embryotoxicity >3.6g/kg, teratogenicity >6400g/kg
NOAEL (oral, drinking water, rat): maternal toxicity <6.7g/kg, fetotoxicity <5.7g/kg, teratogenicity >6.7g/kg
NOAEL (oral, drinking water, rabbit): maternal toxicity <14.2g/kg, teratogenicity >14.2g/kg
NOAEL (oral, liquid diet, rat): maternal toxicity 8.2g/kg, developmental toxicity =5.2g/kg

Dose descriptor:

NOAEL

5 200 mg/kg bw/day

Dose descriptor:

NOAEC

39 000 mg/m³

Animal data

Pregnant female rats were exposed to ethanol by inhalation at concentrations of 10000, 16000, or 20000ppm in a chamber for 7 hours per day on gestation days 1 -19. On day 20 the animals were euthanized and their fetuses examined. There was no definite increase in malformations at any level of ethanol exposure, although the incidence in the 20000ppm group was of borderline significance. There was clear maternal toxicity evident at the highest dose (narcosis, food intake reduction). A NOAEL for maternal toxicity of 16,000ppm was established (30,400mg/m3 ethanol, 58,6000mg/m3 ethyl acetate equivalent theoretical concentration) and a NOAEL for teratogenicity of 20,000ppm (38,000mg/m3 ethanol) was established.

Pregnant female mice were exposed to ethanol at 2200, 3600, 5000, 6400 and 7800 mg/kg/day by gavage from day 8 to 14 of gestation. Dams were sacrificed on day 18 and the content of the uterus and litters examined. Maternal mice treated at concentrations of 3600mg/kg ethanol and higher were lethargic and showed staggered gait and/or laboured breathing. Lethality in maternal animals increased dose dependently from 3600 mg/kg up to the highest dose group resulting in the death of all animals. At 5000 mg/kg, resorption of litters were increased and live foetuses/litters were decreased. This was not apparent in the one litter at 6400 mg/kg. No other fetal effects were seen. This study determined a NOAEL of 3600mg/kg ethanol for embryotoxicity and 2200mg/kg for maternal toxicity. No teratogenic effects were seen even at the highest dose tested.

Pregnant mice were fed a liquid diet containing 17%, 25%, or 30% ethanol-derived calories from day 4 to day 9 of gestation. Dams were sacrificed on day 18 of gestation. Ethanol treatment in dams did not induce any increase in mortality or change in weight gain with respect to controls. The incidence of fetal resorptions and congenital malformations increased in a dose-related manner with significant effects in the groups treated with 25% and 30% ethanol-derived calorie diets, but no significant adverse effects were seen in the low dose group (estimated daily dose ~13-14g/day).

Female CBA and CH3 mice were exposed to liquid ethanol diet providing between 15%, and 25% ethanol derived calories for a period of at least 30 days before mating untreated males, and throughout gestation. Females were killed on day 18 of gestation and offspring examined for skeletal and soft tissue abnormalities. The CBA mice were more sensitive: at 15% and higher doses an increase in resorptions and significant number of abnormalities were noted. However, the high doses used in this study and the fact that no NOAEL was established makes it of limited use in predicting the effects from non-oral consumption of ethanol.

Pregnant rats were exposed to 15% ethanol in drinking water from day 6 through 15 of gestation. Animals were sacrificed on day 21 of gestation and their litters examined for resorption, number, vitality, weight, size, sex, cleft palate and external alterations of fetuses. Mean consumption of food and liquid by rats given ethanol was significantly less that that of control rats during the experimental period. As a result, mean gain in body weight of the exposed rats was also significantly less between days 6 and 16 of gestation. Ethanol ingestion did not affect fetal survival adversely, but mean fetal body weight was significantly less than that of the control litters. No malformed fetuses were found in the experimental litters. Some skeletal variants consisting of unfused bones of the skull and cervical vertebra with missing centra occurred in the ethanol litters at an incidence significantly greater than in the control litters. It was suggested this was an expected manifestation of the decreased fetal body weight observed.

Pregnant female CD-1 mice were exposed to 15% ethanol in drinking water from day 6 through 15 of gestation. Animals were sacrificed on day 18 of gestation and their litters examined for resorption, number, vitality, weight, size, sex, cleft palate and external alterations of fetuses. Maternal body weight gain reflected the decreased consumption of food and liquid in ethanol treated mice. The incidence of exencephaly, open eye, and cleft palate did not differ significantly from control values. Skeletal malformations were not detected but the incidence of several minor skeletal variants e.g. delayed ossification of the centra of cervical vertebra, non-fused sternebrae and delayed ossification of sternebrae was significantly increased among the litters of mice ingesting ethanol. It was suggested that this increase was an expected manifestation of the decreased fetal body weight observed.

Pregnant female New Zealand rabbits were exposed to 15% ethanol in drinking water from day 6 through 18 of gestation. Animals were sacrificed on day 29 of gestation and their litters examined for resorption, number, vitality, weight, size, sex, cleft palate and external alterations of fetuses. There was an increase in resorptions, primarily due to the complete resorption of two litters in the ethanol group which was attributed to the reduction of liquid intake and loss of weight observed in this group. Fetal body measurements and the number of malformed fetuses were comparable between the control and experimental litters.

Groups of female Sprague-Dawley rats were given liquid diets with 15, 25, or 36% ethanol-derived calories (E), or without ethanol (pair-fed isocaloric (PF) or ad libitum control) for 3 weeks prior to mating, and throughout 21 days of gestation. Prenatal ethanol exposure at 36% ethanol-derived calories (E36, about 10.4 g ethanol/kg bw/day) decreased fetal body weight and length, and skeletal ossification, compared with pair-fed (PF36) and ad libitum controls at GD21. Significant effects on ossification, but not body weight or length, were seen at E25 (corresponding to a dose of about 8.2 g ethanol/kg bw/day), compared to PF25 isocaloric controls. No significant effects on fetal growth or ossification were seen in the E15 group (a dose of about 5.2 g ethanol/kg bw/day from 3 weeks prior to mating to GD 21) compared to the PF15 isocaloric controls. A delay in the development of body weight and skeletal ossification was seen in the ethanol-treated (E25 and E36) fetuses on GD21, compared to the ab libitum controls.

In a study to determine whether low-to-moderate ethanol (EtOH) oral consumption throughout pregnancy in the rat alters maternal mammary gland morphology and milk protein levels, Sprague-Dawley rats were fed an ad libitum liquid diet ~6% vol/vol EtOH throughout pregnancy. Mammary gland weight was unaltered by EtOH, and stereological analysis showed no differences in gland structure compared with control. No effects on hormone levels were seen. Although there were no significant changes in mammary gland gene expression at the RNA level, protein levels of -lactalbumin were increased and whey acidic protein were decreased by EtOH. Offspring of EtOH-fed dams consumed less milk than controls in the lactational period; however, this did not alter their early postnatal growth. Overall, it appears that prenatal EtOH exposure at a dose of around 3.85g/kg does not significantly alter mammary gland development but may alter the composition of the various proteins found within the milk but not sufficiently to affect pup growth.

A study was performed on baboons to assess he impact of binge drinking during pregnancy. The animals were exposed three times by gavage to doses of 1.8/kg ethanol to produce peak blood ethanol levels of 80mg/dL. Doppler ultrasonography was used for longitudinal assessment of fetal biometric parameters and fetal cardiovascular indices at different time points following exposure. Temporary cardiovascular effects were seen but these did not persist until the end of pregnancy. Resultant growth retardation was seen in the ethanol exposed animals (5 -10% compared to controls) manifest as a reduction in head and abdominal circumference but not in terms of femur length. The study provides support for the impact of binge drinking/heavy alcohol consumption on fetal growth but the single, large dose cannot be used to support effects at lower doses or exposure by routes other than deliberate oral consumption (Tobiasz, 2019).

Conclusion from animal data: Ethanol clearly can cause developmental toxicity. However, the doses required to cause such effects in animals are exceeding high compared to doses normally used to assess the hazards of chemical substances. Such doses are clearly also associated with maternal toxicity and are likely to cause significant disturbance of homeostasis, e.g. through nutritional effects. In addition, the blood ethanol concentrations required to cause adverse developmental consequences, known as 'foetal alcohol syndrome' in humans, have been found to be in the range commonly found in alcoholics (150-200 mg/100 ml) and higher (350-800 mg/100 ml). Adverse effects similar to those reported for humans have been induced in rats by large doses of ethanol that result in similar blood ethanol concentrations. However, inhalation exposure of up to 20,000 ppm ethanol did not cause fertility, developmental or neurotoxic effects. Blood ethanol concentrations following the 0, 10,000, 16,000 and 20,000 ppm ethanol inhalation exposures were 0, 3, 50 and 180 mg/100 ml respectively. Ethanol does not appear to cause developmental toxicity up to the maximum dose recommended in guideline studies.

Human data

The only data available is from the consumption of alcoholic beverages and care is required in the extrapolation of the results from such studies to the hazards from exposure by other routes than deliberate oral consumption. Fetal Alcohol Syndrome is know to occur amongst pregnant alcoholics, but such levels of exposure are irrelevant to the assessment of the hazards of ethanol from any non-consumption use. A limited number of studies with potential for extrapolation are summarised in the dossier and here.

In a meta analysis of fifteen studies, no association was found between maternal gestational drinking and the risk of cryptorchidism (OR = 0.97, 95% CI: 0.87–1.07) (Zhang, 2015).

In an epidemiology study designed to assess the foetal and neonatal outcomes in women reporting social intake of low or very low alcohol levels during pregnancy, 1919 cases of pregnant women in Korea were followed in a prospective cohort study. Alcohol consumption was assessed by questionnaire during the first trimester to be an average of 7.6g/week (0.09–47.5g) equivalent to 0 -6 drinks/week. The rates of congenital malformations and chromosomal abnormalities were similar in the exposed and control abstinent groups (p > 0.05). Low-to-very low levels of alcohol ingestion during pregnancy do not appear to be associated with adverse maternal or foetal outcomes. Assuming not less than on drink is consumed at a time, this indicates a no effect level of at least 7.6g (Han (2012).

In a large prospective cohort study, no adverse link was found between alcohol consumption during pregnancy and preterm birth or small birth weight. In fact, there was a significant reduction in the risk of pre-term birth even after correcting for potential confounding factors (Pfinder, 2013).

In a study designed to examined the association of maternal alcohol consumption during pregnancy with the risk of pre-term birth and small for gestational age outcomes in Japanese women, consumption of >1g ethanol was just significantly associated with a risk of preterm birth (95% CI = 1.00 to 5.80) although the test for trend was significant. No other parameters (small for gestational age or birthweight) were significantly affected. The meaningfulness of the measure make these results difficult to interpret since the results must be averaged out over many days and no beverage consumed as a single unit only contains 1g of alcohol (Miyake, 2014)

In a study to assess the association between the risks of spina bifida in relation to cigarette, alcohol, and caffeine consumption by women during the first month of pregnancy, high alcohol intake (average ≥4 drinks/day) was not associated with increased risk of Spinabifida (Benedum, 2013).

In a study to investigate the perinatal outcomes in pregnant women who habitually consumed alcohol during pregnancies managed at a major perinatal centre, regular consumption of >10g/day of alcohol in the form of alcoholic beverages was not associated with any adverse perinatal outcomes (Ikeda, 2015).

A prospective cohort study in the SW of the UK examined the association between maternal drinking during pregnancy and trajectories of offspring weight and height from birth to 10 years of age. The study found that in all categories up to women who drink 1 -2 drinks/day and do not binge drink had no adverse findings in the weight and height of their offspring. Even for heavy drinkers (1 -2 drinks/day early pregnacy, and either >3 drinks/day mid pregnancy and/or at least one binge drinking session), whilst there was evidence of a weight and height reduction at birth, this disappeared by the 2nd birthday (O'Keeffe, 2015).

A sub-group of a large prospective cohort study was follwed to investigate the possible effects of weekly low to moderate maternal alcohol consumption and binge drinking episodes in early pregnancy on choice reaction time (CRT) and information processing time (IPT) in children at 5 years of age. After adjustment for a wide range of potential confounders, there were no significant effects of average weekly maternal alcohol consumption during pregnancy on CRT or IPT. There was, however, an indication of slower CRT associated with binge drinking episodes in gestational weeks 1–4 (p=0.05). Care is needed interpreting the latter as this change whilst significant is quite small and represents one observation out of many (random effect?) The band of ''binge drinking' is very wide and can include high consumption individuals that could skew the results across the whole observation band. (Kilburn, 2015).

An epidemiology study to examine the relationship between fetal growth and maternal alcohol consumption, particularly light consumption, before and during early pregnancy found no evidence for adverse outcomes from the light consumption of alcoholic beverages (1 -2 units per week.) In fact the authors reported no relationship between light alcohol consumption during early pregnancy and the subsequent mean birth weight, preterm delivery (%), small-for-gestational age (%) and mean neonatal head circumference (Reynolds, 2019)

A meta-analysis of 24 studies reporting alcohol exposure during pregnancy and miscarriage concluded that alcohol consumption during pregnancy increases the risk of miscarriage by between 12 -28%. More quantitatively, for alcohol use of five or fewer drinks per week, each additional drink per week was associated with a 6% increase in risk of miscarriage (95% CI 1% to 10% increased risk). Extrapolating such a result to other non-drinking forms of alcohol consumption is not possible with any reliability due the marginal effects seen in the context of the weaknesses of the underlying studies estimates of alcohol consumption (Sundermann, 2019).

A case controlled epidemiology study to assess the impact of alcohol consumption on babies gestational size found no significant impact from daily alcohol consumption of up to or above 8g/day. In fact, there was some evidence that light alcohol consumption actually significantly reduced the risk of small for gestational age newborn compared to abstainers (adjusted OR=0.62 (95%CI 0.43-0.88). Smoking, preeclampsia, intrauterine growth retardation, weight gain during pregnancy and body mass index were all found to have a highly significant impact on gestational size and therefore potential confounding factors that need to be controlled for. The authors notde that women asked directly about alcohol consumption during pregnancy tend to under-declare their intake. This would tend to overestimate any effects noted with alcohol consumption. This study provided good evidence that light alcohol consumption does not affect the gestational size of the offspring of pregnant women (Martinez-Galiano, 2019).

These studies suggest that there is no hazard associated with exposure to alcoholic beverages from light to moderate drinking from which it can be extrapolated that there is no hazard from exposure to ethanol by inhalation or dermal exposure. Indeed, a review of alcohol drinking during pregnancy concluded that drinking 20g ethanol twice per week is acceptable (Anonymous, 2001). . It is not possible to achieve the blood ethanol concentrations by inhalation that would produce concentrations that would result from a 20g oral exposure.

Overall, it can be concluded that adverse effects from the effects of ethanol treatment are only seen at very high doses only relevant to deliberate and repeated oral consumption of ethanol. The most important studies are the 2-generation study which shows a NOAEL of 13.8g/kg and the inhalation studies that show a NOAEC of 16000ppm (the maximum tested exposure, which is close to or exceeding 50% of the lower explosive limit.) On this basis, it can be concluded that it is impossible to reach the doses of ethanol required to produce any sort of adverse reproductive response other than by repeated oral consumption of large amounts of ethanol, doses normally only associated with problem drinking, and therefore classification for reproductive or developmental toxicity in the context of a chemical substance is not appropriate or warranted.