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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:
test procedure in accordance with national standard methods

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
Principles of method if other than guideline:
Other information: Since there were no significant effects on Task 2 fertility and reproductive performance endpoints, Task 4 was conducted with only the control and 20,700 mg/kg/day exposure groups.
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.

The direct rapid in vivo hydrolysis of ethyl acetate to ethanol allows the use of this study for ethyl acetate hazard assessment for systemic end points such as fertility.

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. Dosing formulations in the main study were found to be within 2% of nominal.
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 / concentrationsopen allclose all
Dose / conc.:
5 other: % v/v in drinking water
Remarks:
Dose calculated as 6900mg/kg/day. Dose only used for F1 generation
Dose / conc.:
10 other: % v/v in drinking water
Remarks:
Dose calculated as 13800mg/kg/day. Dose only used for F1 generation
Dose / conc.:
15 other: % v/v in drinking water
Remarks:
Dose calculated as 20700mg/kg/day. Dose used for both F1 and F2 generation
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%

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. The following parameters were examined in F1 offspring: Litter data, weight gain.
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:
Yes
Offspring viability indices:
Proportion of pups born alive.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality in P animals is reported but not discussed. No mortality in dose range finder up to 15% ethanol for 14 days.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
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. Changes not regarded as significant. No effects seen on postpartum dam body weights.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
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%.
Organ weight findings including organ / body weight ratios:
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
Description (incidence and severity):
Fertility indices were 97, 100, 100 and 94% in the controls and 5%, 10%, 15% ethanol groups respectively. Other reproductive performance indices e.g. gestation index, changes in lactation and changes in oestrous cycles were not studied.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
20 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed in parameters studied at all doses.

Results: P1 (second parental generation)

General toxicity (P1)

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Postpartum dam weights reduced by 9% in dosed animals following delivery of a single litter

Reproductive function / performance (P1)

Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Significant reduction in sperm motility in high dose animals (-32%). No sperm abnormalities observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
The fertility/mating index of the 15% group was 65%/70% compared to 85%/90% in the control - this was not noted as statistically significant.

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
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:
no effects observed
Description (incidence and severity):
Not influenced by treatment
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 males from the 15% group at adulthood had decreased bodyweight (-10%) and and decreased weight of testis (-6%) and epididymides (-8%) and seminal vesicles (-18%). These reductions became unsignificant after adjusting for body weight, when relative liver and kidney/adrenal weights were increased (+11% and +12% respectively) The latter changes were also seen in females (+13%/+11%).
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

The F1 offspring of the 15% ethanol pairs had significantly fewer live pups per litter (22% reduction). There was a statistically significant 16% reduction in male pups at 5% but no significant differences were seen in the mid dose group. Live pup weight was reduced in the low dose group in both sexes, which remained signiificant after adjustment for the females only. The lack of effects in the mid dose group suggests that the changes seen in the low dose group were not treatment related.

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).

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

- Vaginal opening or preputial separation: Not studied.
- Anogenital distance: Not measured.

No effect on sex ratio, litters per pair (range 4.5 - 4.9) or proportion of pups born alive.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
13 800 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: pups per litter were observed and significant changes to sperm motility.

Results: F2 generation

General toxicity (F2)

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Adjusted but not unadjusted live pup weight reduced in dosed animals (reduction 7%).

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
< 20 700 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Lower live pup weight observed at the 15% dose studied. Lower doses not examined.

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.

 

The reproductive effect noted in Task 2 was a reduction in the number of live pups per litter (male, female, or combined) in a group receiving 20,700 mg/kg/day.  No effects on this parameter were noted at 13,800 mg/kg/day.  The effects noted in the male and female offspring exposed to 20,700 mg/kg/day during Task 4 included reduced body weights at weaning and at study termination, reduced fertility and mating index, reduced pup (F2) weights (after adjusting for litter size), reduced body weight for dams (F1 female rats), reduced parental (F1) body weights at necropsy, and increased relative liver, kidney and adrenal weights in the F1 animals.

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-generational 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.

Synopsis

The lowest NOAEL seen across the P, F1 and F2 generations was 10% ethanol, which was equivalent to: 13800mg/kg/day.