Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with water and ethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to regard it as reliable for use in hazard assessment.Limited experimental detail provided.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

untreated females, rationale for doses selected not provided

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding laboratory
- Age at study initiation: 2 Months old at acquisition
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 1 degree C
- Humidity: 45 +/- 5%
- Photoperiod: 12 hr:12 hr light:dark cycle

Route of administration:

oral: gavage

Vehicle:

other: distilled water only

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Exposure period: 9 weeks
Premating exposure period (males): 9 weeks
Food and water was removed at the time of first intubation at 9am and returned at 5pm.

Frequency of treatment:

twice per day

Details on study schedule:

After treatment, each male was paired with 2 untreated 75-90 day old females. Mating was confirmed by presence of plugs. Females were allowed to deliver their litter.

Remarks:

Doses / Concentrations:
0, 2, 3 g/kg twice daily, total dose 0, 4, 6g/kg/day
Basis:
other: nominal dose

No. of animals per sex per dose:

groups of 20.

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

Groups receiving 2 g/kg or 0 g/kg were paired fed with those receiving 3 g/kg and a fourth group received no gavage treatment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of 10 pups per female.

PARAMETERS EXAMINED
The following parameters were examined in offspring: Culled pups were subjected to brain and adrenal gland weight measurements. Offspring were weighed at 7, 14 and 21 days. At 7 days old, three males and 3 females from each of 6 litters were culled and their brain and adrenal gland weights determined. At 21 days, this was repeated with inclusion of more organ weights. Blood alcohol levels were determined after breeding was determined at 1,2,3 & 5 hours after dosing.

Statistics:

ANOVA and Duncan's Multiple Range tests on parametric data; Chi-square on non-parametric data.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no adverse effects on male reproductive performance and female fecundity was not affected.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Dose descriptor:

NOAEL

Effect level:

> 6 000 mg/kg bw/day (actual dose received)

Sex:

male/female

VIABILITY (OFFSPRING)
Litter sizes and birth weights were not affected. Litter sizes and birth weights were not affected by paternal ethanol intake at either dose.

BODY WEIGHT (OFFSPRING)
There was a significantly higher number of female runts (bodyweight <5.5 g) in the groups sired by rats exposed to ethanol. There was also a significantly higher number of male runts in the groups sired by rats exposed to ethanol. In both cases, these were only statistically significant in comparison to the vehicle treated controls. There was a slight difference between the untreated control animals as the high dose group but this was not statistically significant. In fact the differences between the two controls reached statistical significance. Ethanol treatment in fathers had no effect on offspring growth rate.

ORGAN WEIGHTS (OFFSPRING)
Ethanol treatment at both levels resulted in a significant increase in absolute and relative adrenal gland weight but not in brain weight in comparison to the vehicle treated controls but not the untreated ones. Organ weights (both absolute and bodyweight relative) were unaffected at 7 days but significant reductions of absolute spleen and heart weight and relative spleen weight were noted at 21 days at the 3g/kg dose level (NOEL=2g/kg) and reported to be statistically significant. It should be noted again that this statistical difference is not apparent in comparison to the untreated controls. Also, the reductions in weight in the mid dose group as reported are even larger, but not reported to be statistically significant.

OTHER FINDINGS (OFFSPRING)
The % of males in litters sired by ethanol treated rats was significantly lower than the % sired by vehicle-treated fathers (p<0.04) although the difference with the non-intubated control was not significant.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

4 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Reduction of spleen and heart weight observed at 21 days in comparison to concurrent vehicle control where effects reported to be statistically significant.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 6 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: in comparison to non treatment control

Reproductive effects observed:

not specified

The percentage of males in treatment groups was reported to be significantly lower than controls but this was thought by the authors to be a chance finding.

Conclusions:

There was no effect on fertility in a group of 20 male rats given 3 g or 2 g/kg ethanol by oral intubation daily for nine weeks, achieving BAL's of 338±15 and 132±5mg/dL, respectively. Other conclusions remain uncertain.

Executive summary:

Male rats were treated with 2 or 3g/kg ethanol twice a day by oral intubation for 9 weeks. At the end of the treatment they were paired with two untreated females each. Pregnant females were allowed to deliver their litter. The brain and adrenal weights were measured in the culled animals at birth, on postnatal day 7, and finally on postnatal day 21 with additional organ weight measurements. Paternal alcohol exposure did not influence litter size, average birth weight per pup or postnatal bodyweights in offspring. However, it induced a significant increase in the number of runts in the highest dose group which suggests an influence of ethanol on individual sperm rather than on entire sperm production. Treatment with ethanol also induced an increase in spleen weight at birth that did not persist, and a decrease in heart and spleen weight at 21 days at the highest dose treated. However, it should be noted that no findings were statistically significantly different from both controls used and that for those end points where the treatment groups caused statistically significant findings, the two control groups also varied significantly. On this basis, the positive findings reported here should be given low weighting.

Reference 2

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A published study containing sufficient details to regard it as reliable for use in hazard assessment. Not a standard study protocol but appears to be well conducted and reported. Limited and very high doses does not allow a NOAEL to be established.

Qualifier:

no guideline available

Principles of method if other than guideline:

Method: other
Study of male mice fertility function and its reversibility following abstinence from treatment

GLP compliance:

not specified

Limit test:

yes

Species:

mouse

Strain:

C57BL

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories (Bar Harbor, ME).
- Age at study initiation: 60 days
- Housing: individually.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet): ad libitum.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 degree C
- Humidity: 45 ± 5%
- Photoperiod: 14 hr:10- hr light:dark cycle.

Route of administration:

oral: feed

Vehicle:

other: liquid diet

Details on mating procedure:

No matings conducted.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 35 or 70 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
5 and 6% in feed
Basis:
nominal in diet

No. of animals per sex per dose:

Group sizes consisted 5 to 12 individuals (treatment and control groups respectively).

Control animals:

other: Vehicle: (Carnation Slender containing sucrose as above).

Details on study design:

Control animals were pair-fed to the diet consumed by the ethanol-treated animals on the previous day.

Statistics:

Wilcoxon tests; parametric data analysed following appropriate transformations and Newman-Keuls multiple range test.

Dose descriptor:

NOAEL

Effect level:

< 5 other: % ethanol in feed

Sex:

male

Basis for effect level:

other: Reproductive effects at the lowest dose tested

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Reproductive effects observed:

not specified

BAL peaked at 166 ± 38 mg% (1660 mg/l, n=15) for the 5% dose and 260 ± 35 mg% (2600 mg/l) for the 6% dose, both at day 34/35.

After treatment with either 5% ethanol or 6% ethanol, testicle weight decreased by 24% and 28%, but the effect was reversible and there was no significant difference between the control and recovery animals (10 weeks no treatment, P > 0.1).

Seminal vesicle/prostate weights decreased by 20%, for those on the 6% diet but the effect was reversible and there was no significant difference between the control and recovery animals (10 weeks no treatment, P > 0.1).

There were significant increased frequencies of germ cell desquamation (480% in the 5% treatment group, 400% in the 6% treatment group) and of inactive seminiferous tubules (186% in the 5% treatment group, 567% in the 6% treatment group).  Improvement in both parameters was noted in the contralateral organs after 10 weeks alcohol abstinence but all remained significantly elevated except for the % inactive tubules which returned to control group levels in the 5% treatment group.  (Note: Germ cell desquamation in 5% treatment recovery group; 95% confidence limit range: 1.2 to 3.2% lumina showing desquamation versus control level of 0.3 to 1.0%).

Quality of spermatogenesis was significantly poorer in tests from both treatment groups compared to their respective controls.  After 10 weeks abstinence, some pathology persisted in animals that had been exposed to ethanol although the differences were not significant.

Caudal epididymal sperm content was not significantly affected by treatment with the 5% ethanol diet but was 6% lower in the animals receiving the 6% diet (p < 0.01).  This difference disappeared following 10 weeks without treatment.

Sperm motility was not significantly affected by treatment with the 5% ethanol diet but in the animals receiving the 6% diet motility was reduced by 85%.  This difference disappeared following 10 weeks without treatment.  Forward progression was reduced in both treatment groups (apparently more so in the lower treatment group).  The difference disappeared in the recovery group that had been receiving 5% ethanol but persisted in the 6% group.

In vitro fertilization of mouse oocytes by epididymal spermatozoa was reduced by 20% in the 5% treatment group and 63% in the 6% treatment group but these differences disappeared in both treatment groups following 10 weeks abstinence.

No information is provided on food or ethanol intake or body weight. If bodyweight is assumed to be 30g and liquid diet intake ~8.5g/animal, then 5% in diet will be equivalent to ~14g/kg/day ethanol.

Conclusions:

No NOAEL established (<5% ethanol diet, <166mg% BAL). However, for persistent effects the NOAEL would appear to be close to 5% ethanol diet.

Executive summary:

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.

Reference 3

Endpoint:

toxicity to reproduction

Remarks:

other: Study of ovarian morphology and function

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Not to a standard protocol. High doses used.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Female rats were studied to assess ovarian morphology and function following treatment with ethanol.

GLP compliance:

not specified

Species:

rat

Strain:

other: Holtzmann

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: aged 20 days at start.

Route of administration:

oral: feed

Vehicle:

other: liquid diet

Details on exposure:

VEHICLE
- Ethanol was supplied in a liquid diet.

Details on mating procedure:

No matings attempted, therefore, no F1 and F2 animals.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 55 days
Premating exposure period (males): none
Premating exposure period (females): 50-55 days

Frequency of treatment:

ad libitum daily

Remarks:

Doses / Concentrations:
2.5% and 5% in feed, estimated 8-12 g/kg/day and 16-20 g/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

8-11 per group.

Control animals:

not specified

Details on study design:

- Dose schedule: Pair-fed controls were used at each dose for P animals with ethanol in diet ad lib for 50-55 days.

Parental animals: Observations and examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

Females were examined daily for vaginal patency. Once patent, vaginal lavages were made daily. Oestrous cycle length but not pattern was determined.

Sperm parameters (parental animals):

Not applicable.

Litter observations:

Litter standardization: Not applicable.
F1 and F2 observations: Not applicable.

Postmortem examinations (parental animals):

Blood was collected.
Ovaries and uterine horns were dissected free from surrounding tissue and weighed to the nearest 0.1mg.
Ovaries, uteri, and vaginae were fixed for histology

Statistics:

ANOVA and Duncan's multiple range test

Dose descriptor:

NOAEL

Effect level:

2.5 other: % in diet

Sex:

female

Basis for effect level:

other: ovarian function parameters

Reproductive effects observed:

not specified

Result: Ovarian function was suppressed at the high dose.

Ovarian function was suppressed in rats that achieved blood alcohol levels of 250 mg/100 ml.
Animals receiving 5% ethanol (but not 2.5%) in liquid diet had longer time to vaginal patency, failed to begin oestrus cycles, showed decreased bodyweight gain, had ovaries containing only a single generation of corpora lutea, had infantile vaginal and uterine epithelium and decreased uterine and ovarian weight.

Conclusions:

Ethanol treatment at 5% concentration, but not 2.5%, suppressed ovarian function.

Executive summary:

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.

Reference 4

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Key details of study reported. Only a single, very high dose which limits usefulness for assessment outside of beverage consumption. Exposure actually sub-acute rather than sub-chronic - could be considered short for such a test.

Principles of method if other than guideline:

Exposure of male and female mice to ethanol in drinking water. Male sperm and female gametes then examined for adverse effects

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

CF-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeding Laboratory of the School of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires, Argentina,
- Age at study initiation: 60 days
- Weight at study initiation: 27-30g
- Housing: 3 or 4 per same sex cage.
- Diet (ad libitum): commercial mouse chow (Alimento “Balanceado Cooperación Rata-Ratón,” from the “Asociación Cooperativas de Alimentos,” S.A. Buenos Aires, Argentina)
- Water (ad libitum): hyperchlorinated tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Photoperiod (hrs dark / hrs light): 14/10

Route of administration:

oral: drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dose and control group diets were adjusted to ensure calories derived from food plus ethanol were the same as control food and tap water only group.

Details on mating procedure:

Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Intake of drinking water monitored daily. Sub group of treated animals sacrificed at end of study for trunk blood analysis of plasma ethanol levels.

Duration of treatment / exposure:

27 days

Frequency of treatment:

Continuous (drinking water)

Remarks:

Doses / Concentrations:
Measured ethanol intake 23g/kg (+/-2.2) males, 21.5g/kg (+/-1.6) females.
Basis:
actual ingested

Remarks:

Doses / Concentrations:
198 -281mg/l.
Basis:
other: Measured ethanol blood plasma concentrations

No. of animals per sex per dose:

6

Control animals:

yes

Positive control:

No

Sperm parameters (parental animals):

Parameters examined - sperm morphology: head morphology, the acrosomal and post-acrosomal regions, cytoplasmic droplet, mid-piece, and flagellum.

Statistics:

Sperm morphology and ovulation rates: Student's t-test. Oocyte frequency: Chi-squared test.

Clinical signs:

not specified

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:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Food and drink intake but not calorific intake reduced.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Head and tail abnormalities significantly increased

Reproductive performance:

not examined

Ethanol-treated females in the oestrous phase had a significantly diminished mean number of ovulated oocytes compared to controls. Immature
oocytes were easily detected by the presence of the central germinal vesicle and rough zona pellucida. Ethanol treated females had a significant higher percentage of activated and degenerated oocytes versus controls.

Dose descriptor:

NOAEC

Effect level:

< 21 500 mg/kg bw/day (nominal)

Based on:

other: estimated dose received

Sex:

male/female

Basis for effect level:

other: Abnormal effects in sperm (head and tail defects). Treated females in the oestrous phase had a significantly diminished mean number of ovulated oocytes and a significant higher percentage of activated and degenerated oocytes versus controls.

Reproductive effects observed:

not specified

Executive summary:

In a study to evaluate morphological sperm and oocyte alterations after sub-acute ethanol intake in the CF-1

outbred mouse strain, ethanol 10% was administered in drinking water over a period of 27 days. Intake was in the range 22 -23g/kg/day. Such doses caused abnormal effects in sperm (head and tail defects) and elevated parthenogenetic activated oocyte frequency was also seen. Ethanol plasma concentrations were in the range 198 -281mg/l. Effects at such high concentrations typical of beverage consumption are difficult to extrapolate to hazards likely to be present from exposure during use of ethanol in the workplace or from the use of ethanol containing consumer products.

Reference 5

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Only six pregnancies examined in treatment group. General toxicity such as ataxia, lethargy and weight loss during the experiment may confound the results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

Females not dosed, low number females mated, complete spermatogenic cycle not covered, no pathology conducted, very high single dose only

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 318 g (males); 259 g (females).
- Housing: Individually housed
- Diet: 'Metrecal' (chocolate or vanilla.)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 degC
- Humidity: 45% +/- 10
- Photoperiod: diurnal light cycle (nocturnal 20:00 to 8:00hrs.)

Route of administration:

oral: feed

Vehicle:

other: liquid diet

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Six treated rats were given a 6% v/v ethanol-containing liquid diet (providing 35% of calories.) This was increased to 10% v/v after 7 days exposure (58% of dietary calories).

Details on mating procedure:

Successful matings and the first day of pregnancy was determined by the presence of vaginal plugs and of spermatozooa in the vaginal smears.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 5 weeks
Premating exposure period (males): 15 days
Premating exposure period (females): no treatment /lab chow and water
Duration of test: 36 days

Frequency of treatment:

continuous

Details on study schedule:

- After treatment each male was placed in a cage with 2 females every night. No food or water was provided during this time. The experiment was continued for 5 weeks, with the males still receiving alcohol during the day. The males were killed on day 36. Blood samples were collected.

Remarks:

Doses / Concentrations:
6 increasing to 10% in diet
Basis:
nominal in diet

No. of animals per sex per dose:

12 males; 25 females.
6 controls

Control animals:

other: Six controls given an isocaloric amount of sucrose.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were killed on day 36. Blood samples were collected.
- Maternal animals: Pregnancies were terminated on day 20 of gestation and litter size and foetal mortality was assessed.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Treated males showed signs of intoxication compared to controls. They showed lower serum glucose (p<0.001) levels.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Treated males showed considerable weight loss compared to controls.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Serum testosterone levels were lower in treated animals than controls (p<0.05).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Treated animals were much less successful at mating; the numbers of successful matings were 6/12 in the treated group and 13/13 in the controls. The number of offspring/litter was greater in the controls (p <0.01). A higher incidence of early resorption was seen in the treated group (p <0.01).

Dose descriptor:

NOAEC

Effect level:

< 6 other: % in diet

Sex:

male

Basis for effect level:

other: signs of intoxication

Dose descriptor:

NOAEC

Effect level:

6 - < 10 other: % in diet

Sex:

male

Basis for effect level:

other: Weight loss and reduction of fertility

The mean fetal weight and crown-rump length were significantly greater in the ethanol group than in the controls (p<0.01 for both). The placental index (relating fetal weight to that of placenta) was markedly reduced in the experimental group (p<0.01).

Reproductive effects observed:

not specified

From graphical data presented in the reference, it is possible to estimate ethanol consumption as being in the range 7.2 to 14.4 g/kg/day.

Conclusions:

Treatment of male parent with ethanol induced some adverse reproductive effects.

Executive summary:

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<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.

Reference 6

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Study incomplete as a comprehensive assessment of effects on male fertility. Route of exposure highly relevant.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

short paternal exposure period; no pathology conducted; number of pregnant females low; male treated rats mated to untreated females.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 400-500g males; 200-300g females
- Housing: Females housed individually.

ENVIRONMENTAL CONDITIONS
- Temperature: 73 +/- 3F.
- Humidity: 40-50%.
- Light/dark cycle (hrs): 12/12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air in chamber

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure was conducted in 0.5m3 chambers with dynamic air flow
- Air change rate: (one air change per minute.)

Details on mating procedure:

After 2 day non-exposure period males were mated individually with untreated virgin females. Mating period 5 days. Mating confirmed by presence of sperm plugs under cages or vaginal smears.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 7 hours /day
Premating exposure period (males): 6 weeks
Premating exposure period (females): none
Postmating exposure period (females): Days 1-20 gestation
Duration of test: see method details

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 10000, 16000ppm
Basis:
nominal conc.

No. of animals per sex per dose:

18/group male
15/group female (pregnant)

Control animals:

yes

Details on study design:

Analytical monitoring: Yes (IR analyser) - exposure found to be within 11% of nominal). Independently cross-checked with charcoal adsorption tubes.

Parental animals: Observations and examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food intake were measured.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes

Litter observations:

Pups were individually weighed weekly for five weeks.

Dose descriptor:

NOAEL

Effect level:

> 16 000 ppm

Sex:

male

Basis for effect level:

other: No effects on fertility at highest dose studied

Reproductive effects observed:

not specified

Result: negative

No effect on weight gain, feed or water intake.  No effect on fertility or litter sizes.  Offspring numbers averaged 14/litter. Previous studies quoted as showing exposures to 10000 and 16000 ppm ethanol typically give rise to blood ethanol concentrations of 30 and 500 mg/l ethanol.  Authors calculate that for rats exposures in excess of 11000 ppm are required to begin accumulating ethanol in the blood and that ethanol is no more toxic by the inhalation route than by other routes.

No data on offspring body weight gain provided but it is presumed that there was no change from controls.

Conclusions:

Ethanol treatment did not affect the fertility of male rats determined by number of litters and litter size

Executive summary:

Male rats were exposed to ethanol in an inhalation chamber at concentrations of 11,000ppm or 16,000ppm for six weeks. After a two day non-exposure period they were mated to untreated females for 5 days. The pregnant females received the same experimental exposure from day 1 -20 of gestation and were then allowed to deliver their litters. Ethanol treatment did not affect fertility or litter sizes.

Reference 7

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A published study containing sufficient details and is sufficiently scientifically rigorous to regard it as reliable for use in hazard assessment. Limited experimental detail provided and whilst similar to protocol, some elements were not include, but route of exposure highly relevant to assessment of a chemical substance.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

. Short duration of paternal exposure; low number of pregnant animals (15); no pathology conducted; male only treated premating.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: >300g males; 176-200g females
- Source: Charles River Breeding Labs
- Housing: Females housed individually for breeding and during gestation.
- Acclimation period: 1-2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 24+/-2C.
- Humidity: 40%.
- Light/dark cycle (hrs): 12/12

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air in chamber

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure was conducted in 0.5m3 chambers with dynamic air flow
- Air change rate: (one air change per minute.)

Details on mating procedure:

After 2 day non-exposure period males were mated individually with untreated virgin females (1:1). Mating period 5 days. Mating confirmed by presence of sperm plugs under cages or vaginal smears.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

IR Analyser Miran 1A (Wilkes/Foxboro Analytical), and verified by charcoal tube samples collected from chamber atmosphere (Analysed by Arthur D. Little, Inc. )

Duration of treatment / exposure:

Exposure period: 7 hours /day
Premating exposure period (males): 6 weeks
Premating exposure period (females): none
Postmating exposure period (females): Days 1-19 of gestation
Duration of test: see method details

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 10000, 16000ppm
Basis:
nominal conc.

No. of animals per sex per dose:

Males 18, Females 15

Control animals:

yes

Details on study design:

Females were allowed to deliver their litters. 4 female and 4 male pups were fostered to untreated dams. Rats were weaned at 25 days of age

Positive control:

N/A

Parental animals: Observations and examinations:

Maternal weights, feed intake and water intake measured on gestation days 0, 7, 14 and 21

Oestrous cyclicity (parental animals):

Yes vaginal smears taken if no plugs detected.

Sperm parameters (parental animals):

not examined

Litter observations:

Offspring weighed on days 7, 14, 21, 28 and 35

Postmortem examinations (parental animals):

No data

Postmortem examinations (offspring):

None. Brain samples were taken for neurochemical examination.

Statistics:

parametric ANOVA and nonparametric m-ranking procedure.

Reproductive indices:

Number pregnant/number mated

Offspring viability indices:

No data

Clinical signs:

not specified

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

See below

Dose descriptor:

NOAEL

Effect level:

>= 16 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: No effects on fertility parameters observed nor maternal/paternal toxicity observed at the highest dose studied

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

See below

Reproductive effects observed:

not specified

Result: negative. No effect on weight gain, feed or water intake in maternal animals. Only maternal feed intake during first week was reduced relative to controls at 16,000ppm level. No effect on fertility.  Fertility was >90% in all groups. No group differences found for litter size, number of dead pups, or length of pregnancy. Offspring numbers averaged 11 -15pups /litter. Gestation averaged 22 days. Offspring survival and weight gain did not differ among groups.

Conclusions:

Ethanol did not affect fertility or induce toxicity in female and male rats under the conditions of this study.

Executive summary:

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.

Reference 8

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not a guideline study and publication does not report whether study performed according to GLP. However, data appears well documented and scientifically acceptable.

Principles of method if other than guideline:

Adult male rats were exposed orally to ethanol beginning at puberty. Reproductive parameters such as sexual behaviour, fertility, sperm parameters (production, morphology, motility and counts) were evaluated.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

other: liquid diet

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

After 55 days of treatment, male rats were placed individually in a wooden cage 5 minutes before the introduction of one adult female (in which behavioural estrus was produced by an intraperitoneal injection of estradiol benzoate 24 hrs earlier). Females were killed on gestation day 9 to assess fertility. Males that did not present copulatory behaviour were paired with receptive females for a maximum of 5 consecutive days.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

55 days (one spermatogenic cycle).

Frequency of treatment:

Daily

Details on study schedule:

Two separate experiments were conducted, but the protocol in each case was the same: male rats (50 days old) were randomly assigned to three groups of ten animals each. Group 1 (ethanol-treated) recieved a liquid diet with 36% of the daily calories derived from ethanol and the remainder from chocolate-flavoured liquid feed (Sustacal(R)); Group 2 (isocaloric control) had sucrose substituted for ethanol; and Group 3 (lab-chow control), recieved commercial lab-chow only.

Remarks:

Doses / Concentrations:
13.6 g/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Ten male rats per group; single ethanol dose, isocaloric control, or lab-chow control

Control animals:

yes, plain diet

other: isocaloric control (sucrose)

Dose descriptor:

LOAEL

Effect level:

ca. 13 600 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: Effects observed on sexual behaviour, fertility, sperm production, motility, and abnormalities, absolute weights of the testis, epididymis, vas deferens and seminal vesicle, testosterone levels, and contractile response in vas deferens

Reproductive effects observed:

not specified

Sexual behaviour was altered in ethanol-treated rats, as shown by the low number (2 of 9 ethanol-treated rats) that reached ejaculation in comparison to 5/10 and 7/10 in the isocaloric and lab-chow control groups, respectively. In addition, there was an increase in the latencies of all sexual behaviour parameters studied in the ethanol-treated animals compared to controls, but only the increased latency for the "first intromission post-ejaculation" reached statistical significance.

A significant reduction in fertility after natural (only 17%) and following artificial insemination in utero (78%) was seen when compared to controls (89 -93%). In addition, a reduction in the number of pregnant females in the ethanol-treated rats compared to controls was seen. Preimplantation loss was significantly increased in the ethanol-treated rats following either natural (83% loss) or artifical insemination (22% loss), compared to controls (6 -11% loss).

Ethanol treatment produced a statistically significant reduction in final body weights compared to controls, and the absolute weight of the testis, epididymis, vas deferens and seminal vesicle in comparison to the lab-chow controls (but no significant change from the isocaloric controls). Relative weights of the reproductive organs were comparable in all groups.

A statistically significant decrease in the daily sperm production per testis, as well as daily sperm count in the caput/corpus and the cauda epididymidis (associated with acceleration of sperm transit time in the cauda epididymis) were seen in ethanol-treated rats compared to controls. Furthermore, a statistically significant reduction in sperm motility (25% in ethanol treated, compared to 87 -92% in controls), and increase in epididymal sperm with morphological abnormalities (including higher incidence of abnormally shaped sperm, and tail and head abnormalities) compared to controls were observed.

Plasma testosterone levels were significantly lower in ethanol-treated rats compared to controls. Finally, a significant reduction of 26 and 38% of the maximum contractile response in the vas deferens of the ethanol-treated rats compared to the isocaloric and lab-chow fed controls, respectively, was seen.

Conclusions:

High daily exposure of male rats to a liquid diet containing ethanol (providing about 13.6 g/kg bw/day) from puberty (total of 55 days exposure) impaired sexual behaviour, and only 22% of these rats reached ejaculation compared to 50 and 70% of the isocaloric and lab-chow controls, respectively.The fertililty of the ethanol-treated animals was significantly reduced compared to controls, particularly after natural mating. Sperm production, count, and motility were significantly decreased in ethanol-treated rats, and there was an increase in abnormally shaped sperm cells. Serum testosterone levels were also reduced in ethanol-treated rats, compared to controls.

The single high oral dose level of ethanol in this study makes it of limited value in the overall hazard assessment of ethanol, particularly where incidental exposure (in situations other than through alcoholic beverages) is likely to involve primarily the inhalation and/or dermal routes. Lower dose levels were not assessed, so no dose-response assessment is possible.

Executive summary:

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 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.

Reference 9

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Not a standard protocol but a no effect level can be established by a relevant route of exposure.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male rats were treated with ethanol and the effect in reproductive organs was assessed by the determination of sex organ weight and secretion of testosterone. Hypothalamic-pituitary-gonadal axis was studied as well.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: adults
- Weight at study initiation: 330-350 g
- Housing: Animals housed together in airtight chambers.
- Diet: Laboratory feed ad libitum during exposure. Feed supplemented with vegetables and peanut bars.
- Water: ad libitum
- Acclimation period: acclimated for 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not described.
- Humidity (%): Not described.
- Air changes (per hr): Not described.
- Photoperiod (hrs dark / hrs light): Not described.

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air in chamber

Details on mating procedure:

No mating in study. Not applicable

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 3 to 4 weeks

Frequency of treatment:

continuous

Details on study schedule:

See further details in study design

Remarks:

Doses / Concentrations:
22, 23, 25, 27 mg/l (approx 11500, 12000, 13000, 14000ppm)
Basis:
nominal conc.

No. of animals per sex per dose:

10-12

Control animals:

yes, concurrent no treatment

Details on study design:

- Treatment: During acclimation, all treated rats were exposed to 22 mg/l ethanol vapour in air. After acclimation, groups of 10-12 rats were exposed to ethanol at 23, 25 or 27 mg/l for up to 3 to 4 weeks. Five exposures were run as separate experiments and concurrent controls. After treatment, animals were immediately weighted then sacrificed and trunk blood collected over ice, the plasma separated and frozen for later anlaysis. Sex organs (testes, seminal vesicles, prostate) were removed and weighed. In one experiment, animals were injected with saline or gonadotropin releasing hormone (GnRH) and sacrificed 10 minutes later.
- Controls: Housed in similar chambers to exposed animals without ethanol vapours present.

Positive control:

N/A

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Blood samples were withdrawn periodically from a tail vein for blood alcohol concentration (BAC) determination.
- Plasma testosterone and luteinizing hormone (LH) were recorded.

Oestrous cyclicity (parental animals):

N/A

Sperm parameters (parental animals):

Weight of testes, seminal vesicles and prostate

Litter observations:

N/A

Postmortem examinations (parental animals):

Sex organs were dissected free of fat and weighed

Postmortem examinations (offspring):

N/A

Statistics:

Dunnett's and Duncan's tests after analysis of variance.

Reproductive indices:

N/A

Offspring viability indices:

N/A

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Food consumption measurement not mentioned

Dose descriptor:

NOAEL

Effect level:

23 mg/L air

Sex:

male

Basis for effect level:

other: Decrease in body weight gain observed at next higher concentration level

Dose descriptor:

LOEL

Effect level:

25 mg/L air

Sex:

male

Basis for effect level:

other: Decrease weight gain observed

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

N/A

Reproductive effects observed:

not specified

BACs of 94 to 127 mg/100ml resulted in smaller increase in bodyweight gain than observed in control animals but was not associated with significant changes in plasma testosterone levels or weights of sex organs.  The maximum BAC observed was 187 mg/100ml.  A BAC of >=180 mg/100 ml was associated with an inhibition of testosterone secretion only in animals that had failed to grow. This was not seen in a second experiment with a similar BAC but in which animals exhibited a small gain in weight.

In the experiment where GnRH was injected, a BAC of 163 mg/100 ml was associated with a marked weight loss and reduction of plasma testosterone levels.  Basal plasma LH levels were comparable in the control and ethanol treated rats and intravenous administration of GnRH produced comparable elevations in LH level in the controls and ethanol treated rats.

NOAEL (male fertility) = 127 mg/100ml BAC

LOAEL (male fertility) = 163 mg/100ml BAC

Conclusions:

Authors' conclusion is that data indicates that in growing animals testosterone secretion appears to be directly related to changes in body weight but not the degree of alcohol exposure. Adequate function of the hypothalamic-pituitary-testicular axis provided normal growth is maintained. Alcohol does not appear to lower pituitary gland sensitivity to GnRH.

Executive summary:

Adult male rats were kept in a chamber providing 22, 23, 25, or 27 mg/l ethanol air concentrations 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.

Reference 10

Endpoint:

toxicity to reproduction

Remarks:

other: Study of female reproductive organs

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Not a standard protocol. Very high single dose used and no NOAEL identified.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Method: other
Female rats were studied to assess the ovarian morphology and function and the effect on other reproductive organs and liver following exposure to ethanol.

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Labs
- Housing: individually caged.

Route of administration:

oral: feed

Vehicle:

other: liquid diet

Details on exposure:

Ethanol fed animals received a liquid diet with ethanol accounting for 36% of total calories. Pair fed isocaloric controls (using dextrimaltose as alternative to ethanol.)
Controls: ad libitum intact and ad libitum oophorectomized (Feed: Lab Blox F4 ex Best Feeds, Oakdale, Pa)

Details on mating procedure:

No mating in study

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period: 49 days (animal age 28-77 days)

Frequency of treatment:

continuous

Remarks:

Doses / Concentrations:
5% in feed, or 35% of calories in feed
Basis:
nominal in diet

No. of animals per sex per dose:

100 divided into 4 groups with unspecified number in each. Source: Charles River Breeding Laboratories, Wilmington, MA.

Control animals:

other: See method details

Parental animals: Observations and examinations:

OTHER:
- Plasma steroids and gonadotropins. All assessed by radioimmunoassay’s with following levels of detection:
- Progesterone 10pg, Estradiol 0.1pg, estrone 0.2pg, corticosterone 20pg, and gonadotropin 4ng/ml. All measurements in duplicate.
- Liver: Enzyme function assessed by measuring serum alkaline phosphatase, gamma glutamyl transpeptidase, glutamic pyruvic and glutamic oxalacetic transaminase activities.
- Blood ethanol: measured using blood samples obtained 1 day before sacrifice between 9-11am and before feeding.

Postmortem examinations (parental animals):

SACRIFICE
- Animals sacrificed by exsanguination, 6-7mls of blood kept for analysis.

GROSS NECROPSY
- Liver, ovaries, uterus and fallopian tubes removed and weighed, preserved in Bouin's solution. Liver assessed for degree of fat, necrosis and inflammation. Ovaries assessed for corpus lutea, corpus hemorrhagica, and numbers/development of Graafian follicles. Uterus/fallopian tubes assessed for thickness of endometrium, type of and secretory activity of the epithelial lining cells and thickness of the muscle wall of each organ. Cervix/vagina examined for thickness of epithelial cell lining and signs of estrogen stimulation.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histological sections stained with hematoxylin and eosin.

Statistics:

Student t-test. "Probable significance" at p<0.05 and "significance" at p<0.01

Dose descriptor:

NOAEL

Effect level:

< 5 other: % in diet

Sex:

female

Basis for effect level:

other: Only single dose studied which induced adverse effects in animals

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Reproductive effects observed:

not specified

Growth: (body weight at sacrifice): ethanol fed: 138 +/-5.3g; isocaloric controls: 161.6+/-3.8g. Significant difference (p0.01). Both groups weighed less than the ad libitum-fed control animals (p0.01).

Anatomy: Livers of alcohol fed animals significantly larger than controls  (p0.01. Treated animals 10.6g+/-0.5, isocaloric controls 6.4g+/-0.3, ad  libitum controls 8.3g+/-0.2).  Ethanol treated animal livers markedly more fatty in appearance.
Ovaries: Marked weight loss in treated animals (p0.01;30.6+/-2.2mg versus 75.5+/-3.9mg for isocaloric controls and 91.4mg+/-0.2mg for ad libitum controls).  Weight loss significant even when corrected for body weight.   Reduction in ovarian mass due to absence of developing follicles, corpus lutea and corpus hemorrhagica.

Histology: Differences in appearance of uterus, cervix and vagina between treated and untreated animals.
Uterus/fallopian tube: Marked weight loss in treated animals (p0.01; 39.0+/-4.1mg versus 180.5+/-18.7mg for isocaloric controls and  306mg+/-15.5mg for ad libitum controls).  Weight loss significant even when corrected for body weight.
Plasma steroid and gonadotropin levels (n=25): Plasma estradiol reduction seen in treated animals (ethanol fed:  27.5+/-1.2pg/ml; isocaloric controls: 33.3+/-1.5pg/ml; ad libitum intact controls: 48.0+/-1.4pg/ml; p0.01. However no statistically significant reduction relative to oophorectomized control (29.8+/-1.6pg/ml). Plasma progesterone reduction seen (ethanol fed: 23.3+/-4.3pg/ml;  isocaloric controls: 54.3+/-7.3pg/ml; ad libitum intact controls:  41.7+/-6.7pg/ml; p0.01).  However no statistically significant reduction relative to oophorectomized control (18.0+/-0.6pg/ml).
Biochemistry: Plasma estrone increase seen (ethanol fed: 156.0+/-26.7pg/ml; isocaloric controls: 114.9+/-13.9pg/ml; ad libitum intact controls: 80.5+/-6.3pg/ml; p0.01); oophorectomized control (48.0+/-5.2pg/ml). Plasma corticosterone levels increased (ethanol fed: 74.0+/-9.0ug/dl; ad  libitum controls: 48.0+/-6.0ug/dl; p0.05). However no statistically significant reduction relative to pair fed controls (78.0+/-9.0ug/dl). Plasma lutenizing hormone levels increased (ethanol fed: 68.7+/-5.7ng/ml; ad libitum controls: 43.5+/-7.0ng/ml; p0.01). However no statistically significant reduction relative to pair fed controls (79.4.0+/-6.8ng/ml).   All significantly less (p0.01) than oophorectomized ad libitum control. Plasma follicle stimulating hormone levels not statistically different but all significantly less (p0.01) than oophorectomized ad libitum control.
Biochemical liver function: Serum glutamic oxalo-acetic-acid-transaminase and serum glutamic pyruvic  transaminase levels increased in treated animals (2.5x control levels).   Alkaline phosphatase 50% greater in treated animals (all p0.01). Gamma glutamyl transpeptidase activity not detected in any controls but reproducibly measured (2.3+/-0.6IU/ml) in ethanol fed animals.

Blood ethanol levels: 110+/-9.0mg/l.  Not detected in any controls.

No information is provided on food or ethanol intake or body weight. If bodyweight is assumed to be 350g and liquid diet intake ~37.5g/animal, then 5% in diet will be equivalent to ~5.4g/kg/day ethanol. An alternative calculation which assumes rat calorific intake/day is 225kcal/day/kg and ethanol calorific value is 7.1kcal/g, would lead to an estimate of 11.4g/kg/day.

Conclusions:

Ethanol treatment induced functional and histological adverse changes in the ovaries of rats.

Executive summary:

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.

Reference 11

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Mortality in P animals is reported but not discussed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights at week 13, 38.4+/-0.6 (5, 10 and 15% ethanol) and 39.6+/-0.6g (control); for females 3.4% lower in at 15% ethanol compared with control at week 13.

No food consumption determined only water consumption: Daily water consumption at week 13, 7.0+/-0.1 g per mouse for controls, 7.1+/-0.2g for 5% group, 6.4+/-0.2g for 10% and 5.3+/-0.2g for 15%.

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

VIABILITY (OFFSPRING)
- Not reported. Litters born to P at 15% ethanol had reduced number of live pups per litter.

BODY WEIGHT (OFFSPRING)
- Litter size and weights were not given. Pups born in final F1 generation of animals exposed to 15% ethanol pre- and post-natally weighed less than controls at birth and days 21 and 74.

SEXUAL MATURATION (OFFSPRING)
- Sex ratios: Not influenced by treatment

ORGAN WEIGHTS (OFFSPRING)
F1 males from the 15% group at adulthood had decreased bodyweight and and decreased weight of testis and epididymides and seminal vesicles. In F2 females, relative liver and kidney/adrenal weights were increased.
- Vaginal opening or preputial separation: Not studied.
- Anogenital distance: Not measured.

GROSS PATHOLOGY (OFFSPRING)
- 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

Result: No observed effect on fertility.

Parental/F1 data: Ethanol treatment had no effect on bodyweights and on the proportion of breeding pairs producing at least 1 litter during the continuous breeding phase or the number of litters per pair. 

Effects on sperm and male reproductive organs: In the F1, 15% ethanol group there was a significantly decreased % motile sperm but no changes in sperm concentration, % abnormal sperm or % tailless sperm. There was a significant decrease in testis, epididymis and seminal vesicle weight but not when adjusted for body weight.

Post natal survival until weaning: Not reported.

Estimated daily intakes were 0, 6.9, 13.8 and 20.7g/kg ethanol.

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.

Reference 12

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Only one sex studied. Dose very high.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

Untreated females, no histopathological examination conducted

Principles of method if other than guideline:

Assessment of ethanol toxicity on males only.

GLP compliance:

not specified

Limit test:

yes

Species:

mouse

Strain:

Swiss Webster

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 75 days old at start of treatment

Route of administration:

oral: feed

Vehicle:

other: liquid diet

Details on mating procedure:

- M/F ratio per cage: 2 untreated females per male
- Length of cohabitation: 4 hours
- Proof of pregnancy: vaginal plugs were treated as evidence of pregnancy.
- Females were allowed to give birth and offspring were counted, weighed, culled and re-weighed at 21 days. Litters were standardized by culling at birth to 8 per dam.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Exposure period: 49 days
Premating exposure period (males): Sequential matings through 7 weeks of exposure
Premating exposure period (females): none
Duration of test: 7 weeks

Frequency of treatment:

ad libitum

Details on study schedule:

Number of generation studies: 1

Remarks:

Doses / Concentrations:
10% and 25% of ethanol-derived calories
Basis:
nominal in diet

No. of animals per sex per dose:

20 males per group

Control animals:

yes

Details on study design:

Parameters assessed: Vital and functional observations were maintained on P and F1 generations.

Oestrous cyclicity (parental animals):

Not studied.

Sperm parameters (parental animals):

Not studied.

Postmortem examinations (parental animals):

Sperm quality, anogenital distance and organs at necropsy were not evaluated.

Statistics:

Chi squared test and analysis of variance

Reproductive indices:

At least 80% for each ethanol concentration at each time point. Fertility was at least as great as in pair-fed controls.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical signs were reported. Mortality was not reported.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Paternal bodyweights were less at 25% ethanol-derived calories than at 10 or 0%. High-dose males consumed less diet. (NB pair-fed controls).

OTHER FINDINGS (PARENTAL ANIMALS)
Changes in lactation, haematology, clinical chemistry, gross pathology, no. of implantations, no of corpora lutea, ovarian primordial follicle count, organ weight change and histopathology were not studied.
- Precoital interval: Not measured.
- Duration of gestation: To term.
- Gestation index: Not given.

Dose descriptor:

NOAEL

Effect level:

10 other: % ethanol derived calories in food

Sex:

male

Basis for effect level:

other: Weight loss at higher 25% dose

VIABILITY (OFFSPRING)
- Viability index: Not measured. Litter size not affected.
- Post natal survival: No mortality reported.

BODY WEIGHT (OFFSPRING)
- Offspring bodyweights were not affected by treatment.
- Postnatal growth: Not affected by day 21.

SEXUAL MATURATION (OFFSPRING)
- Vaginal opening: Not studied.
- Anogenital distance: Not studied.

ORGAN WEIGHTS (OFFSPRING)
Not studied.

GROSS PATHOLOGY (OFFSPRING)
Not studied.

OTHER FINDINGS (OFFSPRING)
- Sex and sex ratios: Not affected by paternal, exposure.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

25 other: % ethanol derived calories in food

Sex:

male/female

Basis for effect level:

other: No effects observed at highest dose studied

Reproductive effects observed:

not specified

No toxic responses were noted in treated males other than decreased bodyweight gain at 25% ethanol-derived calories in diet.

Fertility over 7 weeks of treatment was not affected. Fertility was at least as great as in pair-fed or standard controls.

No adverse effects on offspring were noted as a function of either level of paternal ethanol treatment or duration of treatment. Litter sizes and weight were not affected by paternal exposure.

Mean alcohol consumption was 21.5 and 13.9g/kg/day for the 25% and 10% ethanol-derived calories, respectively.

Conclusions:

Paternal consumption of a diet providing 10% or 25% ethanol-derived calories for 7 weeks did not affect fertility or induce changes to litter parameters studied.

Executive summary:

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.

Reference 13

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study containing sufficient details to regard it as reliable for use in hazard assessment. Limited experimental detail provided. Dose levels very high and no clear no effect level established.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

Females were untreated, number of pregnant females low, no histopathology conducted

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, MI but test animals bred in laboratory.
- Age at study initiation: 70 days
- Diet: Laboratory feed ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 degree C
- Humidity: 45 ± 5%
- Photoperiod: 12 hr light/12 hrdark cycle.

Route of administration:

oral: gavage

Vehicle:

water

Details on mating procedure:

Males were bred in once after 3 weeks exposure and twice after 9 weeks of exposure to 75-90 day old females for 2 weeks or until sperm plugs were observed. Males were intubated throughout the mating period. Females with plugs were separated and housed individually. At 20-days gestation, females from the 3-week exposure breeding and one from the 9 week exposure breeding were killed and their foetuses counted, sexed and weighed. The second female from the 9 week breeding was allowed to deliver its litter, which was similarly assessed.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Premating exposure period (males): 3 and 9 weeks
Premating exposure period (females): not treated

Frequency of treatment:

daily

Details on study schedule:

Number of generation studies: 1

Remarks:

Doses / Concentrations:
2.5 and 5.0g/kg
Basis:
actual ingested

No. of animals per sex per dose:

18-26 per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Controls: Vehicle (distilled water without ethanol) and non-intubated controls to evaluate the effects of intubation stress. Animals in the low and control treated groups were pair-fed to the high dose group animals.

Statistics:

Chi-square, ANOVA and Duncan Multiple Range Tests were used.

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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:

effects observed, treatment-related

Dose descriptor:

NOAEL

Effect level:

> 5 000 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: fertility

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: fecundity, but only when data from both breeding periods combined.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Dose descriptor:

NOEL

Generation:

F1

Effect level:

< 2 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Increase in fetal weight observed at both 2.5g/kg and 5g/kg

Reproductive effects observed:

not specified

There did not appear to by any treatment related effects on resorptions and litter size. The mean BAC’s for the two ethanol dose groups was 248 ± 14 mg% (5g/kg) and 155 ± 9 mg% (2.5g/kg). There was a treatment related increase in the number of male foetuses in the 3 week breeding at 5g/kg, but the effect was not repeated at the 9 week breedings. At the 3 and 9 week breedings there was a significant dose related increase in fetal weights.  There was also a significant increase in placental weights, but only at the 9 week breedings. Significantly more males were sired by 5g/kg ethanol treated males than controls in the first breeding.

There were no treatment related effects on newborns sired by alcohol treated males. Male fertility and litter size was not affected by treatment.  Fecundity was not reduced in individual breedings but was significantly reduced at the 5 g/kg dose level if the breeding periods were combined.  There were no differences between the two control groups. At all breedings the concurrent pair-fed control males weighed significantly less than ad lib controls. 

Conclusions:

There was no significant effect on male fertility. Female fecundity was reduced at the high dose level when all breedings were combined but litter size was not decreased nor were resorptions significantly increased. 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.

Executive summary:

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.

Reference 14

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A published study which contains sufficient experimental detail to be able to judge it as reliable. Limited experimental detail provided.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

, untreated males, no dose related effect assessed but exposure, low number of females used, single dose.

Principles of method if other than guideline:

Method: other

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

other: Holtzmann

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: age 20 days. 45-55g.
- 14hr artifical light/day

Route of administration:

oral: feed

Vehicle:

other: liquid diet

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 hr. Mating started on the day of proestrous.
- Proof of pregnancy: sperm in vaginal smear.
- Any other deviations from standard protocol: Study ended with delivery of F1 pups

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Exposure period (females): 8 or 16 weeks before mating
Premating exposure period (males): no treatment . Some males had access to ethanol diet overnight during mating hours in female cage.

Frequency of treatment:

ad libitum daily

Details on study schedule:

Dosing ended after mating.
Rats were allowed to deliver their pups.

Remarks:

Doses / Concentrations:
5% in liquid feed
Basis:
nominal in diet

No. of animals per sex per dose:

10 per group in 5% ethanol groups
6 in pair-fed control in isocaloric diet
7 in standard chow ad libitum

Control animals:

yes

Details on study design:

- Other: Two Control groups were used, one receiving standard diet the other pair-fed with 5% ethanol in diet.
- 16weeks after start of treatment 3 females at vaginal estrus were sacrified and ovaries and uterine horns histologically examined.

Oestrous cyclicity (parental animals):

Oestrous cycle length and pattern was recorded.

Litter observations:

Body weight of the pups was determined

Statistics:

Statistical test was one-way ANOVA.

Reproductive indices:

At least 80% for each ethanol concentration at each time point. Fertility was at least as great as in pair-fed controls.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No clinical signs or mortality were reported.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Bodyweight: Maternal bodyweights were measured but not reported. Offspring bodyweights were not affected by treatment.
- Food/water consumption: Not reported but must have been recorded.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Administration of 5% for 16 weeks, not 8 weeks, increased oestrus cycle length and irregularity. Age to vaginal patency was increased by both regimens. Average age of vaginal patency was 72-77 days in both groups and significantly older than in control groups (41-58 days).
No abnormalities of ovaries or uteri were found. 

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not studied.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Changes in organ weights not studied.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Changes in gross pathology not studied.

HISTOPATHOLOGY (PARENTAL ANIMALS)
All ovaries and uteri examined were normal.

OTHER FINDINGS (PARENTAL ANIMALS)
Changes in lactation, haematology, clinical chemistry, no. of implantations, no.of corpora lutea, ovarian primordial follicle count: Not studied.
- Precoital interval: Not measured.
- Duration of gestation: Not reported.
- Gestation index: All females delivered live litters.

Dose descriptor:

NOAEL

Effect level:

< 5 other: % in liquid feed

Sex:

female

Basis for effect level:

other: oestrous cycle changes

VIABILITY (OFFSPRING)
- Viability index: Not measured.
- Post natal survival: No mortality reported.

CLINICAL SIGNS (OFFSPRING)
- Effects on offspring: Not studied.

BODY WEIGHT (OFFSPRING)
- Postnatal growth: Not studied.

SEXUAL MATURATION (OFFSPRING)
- Sex and sex ratios: Not given.
- Vaginal opening: Not studied
- Anogenital distance: Not studied.

ORGAN WEIGHTS (OFFSPRING)
Not studied.

GROSS PATHOLOGY (OFFSPRING)
Not studied.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 other: % in liquid feed

Sex:

male/female

Basis for effect level:

other: no adverse effects observed in litter parameters observed

Reproductive effects observed:

not specified

Result: Fertility not affected although oestrous cycle length was prolonged and irregular.

Average age of vaginal patency was 72-77 days in both groups of ethanol-treated rats versus 41-58 days in controls.

Estimated ethanol reported to be in the range 14 -21g/kg/day. Whilst serum ethanol levels were not reported in this study, the authors referred to a similar study using the same feed and dosing which produced average serum ethanol levels of 249mg% (+/-57).

Conclusions:

No adverse effect on fertility, litter size or neonatal bodyweight was detected. Irregular cycles and longer oestrous cycles were noted in rats fed for 16 weeks but not after 8 weeks with 8 weeks recovery period

Executive summary:

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.

Effect on fertility: via oral route

Dose descriptor:

NOAEL

13 800 mg/kg bw/day

Effect on fertility: via inhalation route

Dose descriptor:

NOAEC

30 400 mg/m³

Additional information

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.

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)

Justification for classification or non-classification

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 toxicity in the context of a chemical substance is not appropriate or warranted.