Ethyl (S)-2-hydroxypropionate

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-03-28 to 1985-10-29

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 guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted according to the NTP toxicology testing scheme "Fertility assessment by continous breeding" designed to determine the reproductive and fertility effects of ethanol.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol

EC Number:

200-578-6

EC Name:

Ethanol

Cas Number:

64-17-5

Molecular formula:

C2H6O

IUPAC Name:

ethanol

Specific details on test material used for the study:

- Name of the test material used: Ethanol
- Purity: 92%
- Source: United States Industries
- Batch no.: AT 180C03-1

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Lab., Kingston, NY, USA)
- Age at study initiation: (P) animals 6 weeks at receipt, 11 weeks at first exposure.
- Fasting period before study: no
- Housing: for the preliminary dose range finding study 4 animals are housed per cage per sex. For the fertility study: for the first week, animals will be housed five per cage per sex. Subsequently, females and males from the same treatment group will be randomly paired and cohabited for 98 days. One breeding pair per cage. Thereafter each male and female are housed individually. Cages used: solid bottom polycarbonate cages with stainless steel wire lids with Ab-Sorb-Dri cage litter.
- Diet (e.g. ad libitum): ad libitum, pelleted feed (NIH-07 open formula rodent chow)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: t least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70 +/- 2 °F
- Humidity (%): not specified
- Air changes (per hr): 12-14
- Photoperiod (hrs dark / hrs light): 5 am to 7 pm

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A predetermined amount of ETOH will be measured, added to a measured amount of deionized/filtered water (v/v) , and stirred at ~300 rpm until the solution is homogeneous. A different ETOH concentration will be required for each dose level. Each concentration will be formulated independently in a quantity sufficient to be used for two weeks of the treatment period. Each formulated water sample will be refrigerated (4 °C) prior to use. For each formulation, the quantity prepared will be based on the assumption that each mouse will consume at most 15 mL per day and water not consumed will be discarded at least weekly.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 98 days
- Proof of pregnancy: litters were proof of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An aliquot of each formulation of ETOH in the drinking water and the control water and bulk chemical will be sent to Midwest Research Institute (Kansas City, HO) prior to onset of exposure of the 14-day preliminary Range-Finding Study and at week 1, 6, 12 and 18 during the 7 + 98 + 21 day Reproduction and Fertility Study for confirmation of dose levels and to certify stability of the bulk chemical.

Duration of treatment / exposure:

Exposure period: 18 weeks (P0), P1
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:

Parental animals:
Mice (11 weeks of age) will be matched by weight and randomly assigned into four treatment groups. Treatment will be given continuously in the drinking water, both for the seven day premating exposure and the 119-day mating trial.

F1:
The offspring obtained between days 98 and 119 from the high dose and control groups will be cohabited for 7 days beginning at 74 + 10 days of age and their reproductive capacity evaluated. Chemical exposure via the drinking water will continue throughout until the breeding pairs are sacrificed. Twenty male offspring (at least two males from each of 10 randomly selected litters if possible) will be randomly paired with 20 female offspring (at least two females from each of 10 randomly selected litters if possible) from the same dose levels as in in the parental generation. Sibling matings will be avoided. One breeding pair will be housed per cage (high dose male with high dose female; control male with control female) for seven days. If the test chemical causes signicant adverse effects in parental breeders, as determined by statistical analysis after 98 days of cohabitation, offspring from all four dose groups will be bred and examined as above. The females will be checked for vaginal plugs daily and the breeding pairs will be separated after seven days. Vaginal cytology will be performed daily for five days on females who do not present a vaginal plug and do not appear pregnant 16 days after the pairs are separated. Both the males and females will be asphyxiated with CO2, necropsied and evaluated. Litters will be evaluated on postnatal day 0 and then killed by decapitation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: %

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

5 other: %

Remarks:

Low-dose and equivalent to a daily intake of 6900 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

10 other: %

Remarks:

Mid-dose and equivalent to a daily intake of 13800 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

15 other:

Remarks:

High-dose and equivalent to a daily intake of 20700 mg/kg bw/day

No. of animals per sex per dose:

Control group: 40
Dose groups: 20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose selection was based on the results obtained from a dose-range finding study. In this study, animals were exposed to doses of 0, 0.94, 1.88. 3.75, 7.5 and 15% ethanol n their drinking water.

Positive control:

n.a.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily cage side inspections werde done to evaluate clinical signs, morbidity and mortality and delivery of pregnant females.

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: For the one week pre-mating exposure, animals will be individually weighed at exposure onset and on day 7. During the 98-day cohabitation period and 21 days thereafter, adult animals were weighed at the beginning and
end of the first week and at the end of the second, fifth, ninth, thirteenth, and eighteenth week. Adult females will also be weighed on the day they deliver a Iitter. Body weights of F1 breeders were recorded at the commencement of cohabitation and at the conclusion pf wekk 1, 2, 3 and 4.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: water consumption was conducted on day 7 of the pre-mating exposure and once in week 1, 2, 5, 9,13 and 18. The water consumption of F1 breeders was recorded each week.

Oestrous cyclicity (parental animals):

n.a.

Sperm parameters (parental animals):

- Epididymal and vas sperm were evaluated for concentration, motility and morphology in F1 males only.

Litter observations:

During the 98-day cohabitation period and 21 days thereafter, the number of litters produced per breeding pair, the number and percent fertile pairs, the number and percent live per litter and mean body weight of live offspring were recorded. In addition, the litters will be counted, sexed and weighed by sex. For the F1 breeding pairs the following was evaluated: number and percent of fertile pairs, litter size, litter weight, number and % live.

Postmortem examinations (parental animals):

Gross pathology:
In the F1 breeder males the following parameters were evaluated: liver weight, kidneys/adrenaIs weight, prostate weight, seminal vesicles with coagulating glands weight, left testis/epididymis weight, right testis weight, right epididymis weight, number
of cauda epididymal sperm per mg cauda tissue and sperm motility and morphology. The prostate, seminal vesicles/coagulating glands, a portionof the medial lobe of the liver, kidneys/adrenals, and the remaining carcass (with top of cranium removed) are preserved in neutral-buffered 10% formalin. The left testis/epididymis and right testis are fixed for 24 hours in Bouin's solution and then transferred to 70% ethanol; the right epididymis (cauda) is used for sperm analysis.
In the F1 females the following parameters were evaluated: liver weight and kidneys/adrenals weight. The ovaries and oviducts are removed and preserved for 24 hours in Bouin's solution and then transferred to 70% ethanol. The uterus and the upper half of vagina are excised and preserved in 10% formalin. A portion of the medial lobe of the liver, the kidneys with attached adrenals and the carcass (with top of cranium removed) also are preserved in 10% formalin.

Histopathological evaluation of the preserved tissues was not performed since the adverse effects observed in the ethanol treated group were likely due to general toxicity in the F1 animals.

Postmortem examinations (offspring):

n.a.

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:

The number of litters produced per breeding pair, the number and percent fertile pairs, and mean body weight of live offspring were recorded.

Offspring viability indices:

The number and percent live per litter. In addition, the litters will be counted, sexed and weighed by sex

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were observed in either male or female mice in any dose group during the dose-range finding study. For the main test no data was provided.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the 14-day dose range finding study no male or female animals died. In the main test one male died in the control group in week 13. In the low dose one femal died during week 13. In the mid-dose group one female died during week 11 and in the high-dose group one male died during week 1, one male during week 3, one male died during week 11 and one female died during week 16.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The percent change in body weight was unaffected by treatment for both males, females and the sexes combined during the dose-range finding study. In the main study, body weights at week 13 were 38.4 +/-0.6 g for all dosage groups and 39.6 +/- 0.6 g for the control group. For females 3.4% lower in at 15% ethanol compared with control at week 13.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Daily water consumption at week 13, 7.0 +/-0.1 g per mouse for controls, 7.1 +/-0.2 g for the 5% dose group, 6.4 +/- 0.2 g for the 10% and 5.3 +/- 0.2g for the 15% dose group

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: 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):

The fertility index were in the control group 97% (38/39), in the low dose group 100% (19/19), in the mid-dose 100% (19/19) and in the high dose group 94% (15/16).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

15 other: % v/v

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse signs of systemic toxicity observed

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of F1 mice in the 15.0% ETOH group were significantly lower than controls at 21 days of age and at 74 + 10 days of age, indicating reduced rate of growth in pups in the 15% ethanol group surviving to the lactational and postweaning periods.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute left testis/epididymis weight, right epididymis weight, and seminal vesicle weight were significantly depressed (p<0.01 or 0.05) in the 15.0% ETOH-treated males relative to controls. Absolute male liver; kidney/adrenal, right testis, and prostate weight were unaffected by treatment. When F1 male organ weights were adjusted for body weight, liver weight, and kidney/adrenal weight were significantly elevated (p<0.01 or 0.05) over controls in the ETOH-treated group. Adjusted left testis/epididymis, right testis, right epididymis, prostate, and seminal vesicle weights were not significantly different from controls.
For F1 female mice absolute liver weight and kidney/adrenal weight were unaffected. When female organ weights were adjusted for body weight, both liver weight and kidney/adrenal weight in the 15% ETOH group were significantly elevated relative to controls

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Tissues from the F1 animals were saved in 10% formalin, but were not submitted for histopathology since most of the adverse effects observed in the ETOH-treated group were likely due to generalized toxicity in the F1 animals.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

F1 parental animals were necropsied three weeks after the 7-day cohabitation period. Sperm assessment indicated a significant reduction (p<0.01 in sperm motility in the 15.0% ETOH group relative to controls, but no other significant effects were observed in sperm concentration, % abnormal sperm or % tailless sperm.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Continuous exposure of the F1 mice to 15% ETOH in the drinking water via their mothers (in utero) and from birth to 74 +/- 10 days of age had no statistically significant effect on the proportion of detected matings or number of fertile F1 pairs, although the mating and fertility indices for the F1 generation exposed to 15% ETOH appeared to be reduced compared to indices for the F1 control pairs. Fertility indices in F1 matings were 85% and 65% in the controls and 15% ethanol groups respectively. Mating and fertility for the F1 control pairs, however appeared to be reduced compared to values previously observed for the F0 control pairs. There was no significant difference between the control and 15% ETOH-exposed F1 pairs in the number of live pups per litter, proportion of pups born alive, sex of pups born alive, or absolute live pup weight (males, females, or sexes combined). When live pup weight (males, females or sexes combined) was adjusted for the number of live and dead pups per litter, the 15% ETOH group was significantly below (p<0.05 or 0.01) the controls.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % v/v

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex ratio was not influenced by the treatment.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

10 other: % v/v

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F2 offspring weighed less as pups than control pups, males, females or both sexes

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

No effects on the sex ratio of born F2 pups were noticed.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

< 15 other: % v/v

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical results during the main study:

The analysis of thedrinking water formulations at 6 week intervals indicated they ranged from 99 to 101% of the nominal concentrations.

Applicant's summary and conclusion

Conclusions:

In conclusion, ethanol in drinking water at concentrations of up to 15% (equivalent to 20700 mg/kg bw/day) had no demonstrable effect on fertility in this two-generation study.

Executive summary:

In a two-generation study conducted for the National Toxicology Program (NTP) of US EPA, ethanol was administered ad libitum to male and female CD1 mice (40 animals per sex in the control group, 20 animals per sex in the treatment groups) at concentrations of 0%, 5%, 10% and 15% in drinking water (equivalent to 0, 6900, 13800 and 20700 mg/kg bw/day). Animals (P0) were continuously treated for 1 week prior to mating and for a 14-week cohabitation period followed by a 21-day holding period when they were separated and housed individually. Since ethanol exerted no significant effect on the mating and fertility of the P0 breeding pairs,litters of the control and high dose group were randomly selected on day 21 for rearing and were housed by sex in groups of two or three mice and maintained on the same drinking water level of ETOH as their parents. At 74 + 10 days of age, male and female mice from different litters within treatment groups were cohabited for 7 days. The pairs were then separated, and the females allowed to deliver their litters. The parental F1 mice were necropsied three weeks after the 7-day cohabitation period.

Ethanol treatment had no effect on bodyweights and on the proportion of breeding pairs of the P0 generation producing at least 1 litter during the continuous breeding phase or the number of litters per pair. The number of litters produced per pair, the proportion of pups born alive and the sex of pups born alive were also unaffected by exposure to ethanol. Fertility indices were 97, 100, 100 and 94% in the controls and 5%, 10%, 15% ethanol dose groups. In the 15% ethanol group, the number of live pups per litter was reduced.

Body weights of F1 mice in the 15% ethanol group were significantly lower than controls at 21 days of age and at 74 + 10 days of age, indicating reduced rate of growth in pups in the high dose group surviving to the lactational and postweaning periods. Fertility indices in F1 matings were 85% and 65% in the controls and 15% ethanol group respectively. Other reproductive performance indices e.g. gestation index, changes in lactation and changes in oestrous cycles were not studied. In the F1 15% ethanol group (necropsied three weeks after the 7-day cohabitation period) there was a significantly decreased %motile sperm but no changes in sperm concentration, % abnormal sperm or % tailless sperm and there was a significant decrease in testis, epididymis and seminal vesicle weight.

For the F2 generation, there was no significant difference between the control and 15% ethanol exposed F1 pairs in the number of live pups per litter, proportion of pups born alive, sex of pups born alive, or absolute live pup weight (males, females, or sexes combined). When live pup weight (males, females or sexes combined) was adjusted for the number of live and dead pups per litter, the 15% dose group was significantly below (p< 0.05 or 0.01) the controls.

Based on the results the NOAEL parental can be considered to be 15% (20700 mg/kg bw/day) and the NOAEL for the F1 can be considered to be 10% (13800 mg/kg bw/day) and the NOAEL of the F2 offspring must be considered to be lower than 15%. Overall, ethanol in drinking water at concentrations up to 15% (equivalent to 20700 mg/kg bw/day) had no demonstrable effect on fertility in this two-generation study.