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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well reported study following NTP Protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report Date:
1985

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
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:
other: NTP Protocol. Fertility assessment by continuous breeding
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethanol
- Analytical purity: 92%
- Lot/batch No.: AT180C03-1
- Storage condition of test material: room temperature and formulations at 4C prior to use.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

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%.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Results: F2 generation

Effect levels (F2)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.