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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Principles of method if other than guideline:
Assessing the potential of ethylene glycol (EG), when administered by gavage during major organogenesis, to cause maternal and developmental toxicity in rabbits, a common non-rodent test animal species.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethane-1,2-diol
EC Number:
203-473-3
EC Name:
Ethane-1,2-diol
Cas Number:
107-21-1
Molecular formula:
C2H6O2
IUPAC Name:
ethylene glycol

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight: The range of actual body weights for females on GD 0 was 2470 to 4460 g
- Housing: Inseminated females were individually housed in stainless steel cages with mesh flooring
- Diet: Certified rabbit chow, ad libitum
- Water: filtered water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 °C
- Humidity (%): 54.1% (range of 26.8 to 81.9%)
- Photoperiod (hrs dark / hrs light): Animal room lights were on from 7 to 19 hr for females and from 7 to 21 hr for males

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Artificially inseminated New Zealand white does were dosed daily with EG in vehicle or with the deionized/distilled water vehicle on the mornings of GD 6-19. Treatment was by gavage using a stainless steel dosing tube. A dose volume of 5 ml/kg body wt was used for all groups. The volume administered was adjusted according to daily body weights. All formulations were 93 -107% of theoretical for predosing analyses/for the postdosing analyses. All formulations were 91.6-109% of the predosing measured concentrations.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
To induce ovulation, females received an intravenous injection of Pregnyl (0.1 ml/kg) immediately prior to insemination. The day of insemination was designated as Gestational Day (GD) 0. Females were assigned to dose groups (23 -24 per group) by stratified randomization on GD 0 so that body weights did not differ among groups within any individual replicate. The study was performed in two replicates (11-12 inseminated females per dose per replicate) with two consecutive breeding days in each replicate. The last breeding date for the first replicate and the first breeding date for the second replicate were 5 weeks apart.
Duration of treatment / exposure:
gestation day 6 to19
Frequency of treatment:
daily
Duration of test:
until gestation day 30
No. of animals per sex per dose:
23 - 24
Control animals:
yes, concurrent no treatment
Details on study design:
The doses chosen were based on preliminary studies with nonpregnant rabbits and information from rodents which indicated developmental effects at much lower doses than maternal effects. Analyses of the dosing formulations indicated that they were homogeneous and stable for at least 3 weeks.

Examinations

Maternal examinations:
Inseminated females were weighed on GD 0, 6-19, 25, and 30. Females were observed and maternal water consumption was measured daily throughout gestation GD 0-30. All surviving inseminated females were killed at scheduled necropsy on GD 30 by iv injection of a euthanasia solution into the marginal ear vein. The maternal liver, kidneys and intact uterus were weighed and corpora lutea counted. Uteri which presented no visible implantation sites were stained with ammonium sulfide (10%) to detect very early resorptions. Maternal kidneys were bisected and fixed in 10% neutral buffered formalin. Kidneys from 10 randomly selected does from the control through the 1000 mg/kg/day EG groups and all the kidneys from the 2000 mg/kg/day EG group as well as kidneys from all does which died during study were sectioned, stained with hematoxylin/eosin and evaluated histologically. The sections were also examined under polarized light for oxalate crystals.
Fetal examinations:
Live fetuses were dissected from the uterus and euthanized. They were weighed, examined for external morphological abnormalities including cleft palate and dissected for visceral examination and determination of sex by a fresh tissue dissection technique. Half of the fetuses were decapitated after dissection: the heads were fixed in Bouin's solution and then examined by a freehand sectioning technique. All fetal carcasses were skinned, cleared, stained with Alcian blue/alizarin red S. and examined for skeletal malformations and variations.
Statistics:
Analyses were performed using the doe or the litter as the experimental unit. General Linear Trend Models procedures were applied for the analysis of variance (ANOVA) of maternal and fetal parameters. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter-derived percentage data and Bartlett's test for homogeneity of variance was performed on all data to be analysed by ANOVA.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clear treatment-related clinical signs of toxicity were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
42.1% mortality (8 of 17 pregnant animals) at 2000 mg/kg/day
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Periodic maternal body weights and weight changes were statistically equivalent across all groups for all intervals evaluated.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Maternal water consumption, expressed as g/animal/day or as g/kg/day was also statistically equivalent across all groups for all intervals, although water consumption appeared slightly increased for all EG-dosed groups during the treatment period but not in a manner related to dose.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry 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):
At scheduled necropsy on GD 30 there were no significant effects of treatment on corrected (for uterine weight) maternal gestational weight change, gravid uterine weight, liver weight, or kidney weight at any dose.
However, maternal absolute kidney weight (but not relative weight) was slightly increased at 2000 mg/kg/day to 106.3% of the control value for the right kidney (left kidney value was 107.6% of control).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At scheduled necropsy on GD 30 there were no significant effects of treatment on corrected (for uterine weight) maternal gestational weight change, gravid uterine weight, liver weight, or kidney weight at any dose.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histologic evaluation of maternal kidneys revealed treatment-related renal lesions only at 2000 mg/kg/day. The lesions were limited to the cortical renal tubules and included intraluminal crystals (appearance consistent with oxalate), epithelial necrosis, and tubular dilatation and degeneration. The most severe findings, crystals (designated "marked") and necrosis, were observed in the does which died on study, but the renal tubular necrosis observed in these animals was not a postmortem event. The cause of death in these animals was determined to be renal failure.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One doe at 2000 mg/kg/day aborted on GD 20 (no litters were aborted at any other doses). Increases in early deliveries and abortion are usually considered indicative of maternal stress in rabbits.
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
The 8 does which died at 2000 mg/kg/day died on GD 9 (one doe) and 11 (two does) and on GD 13, 14, 19, 21 and 25 (one doe each).
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
2000 mg/kg/day: increase in early deliveries with three at this dose (and one each at all other doses)
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
Pregnancy rate was high and equivalent across all groups from 0 - 1000 mg/kg/day (95.5, 95.7, 91.3, and 95.2%, respectively); pregnancy rate was slightly lower (8 1.8%) at 2000 mg/kg/day.
Other effects:
not specified
Description (incidence and severity):
There were no treatment-related effects on any gestational parameters, including no effects on number of ovarian corpora lutea, on total non-live or live implantation sites per litter, or on pre- or post-implantation loss. There was a statistically significant increase in the number of corpora lutea at 500 mg/kg/day associated, as expected, with a slight increase in the number of implantation sites/litter and a slight increase in live litter size. This finding was not observed at higher doses and is considered most likely due to biologic variation. The values at 500 mg/kg/day are still well within historical control values for these parameters.
Details on maternal toxic effects:
Details on maternal toxic effects:
Maternal toxicity was observed at 2000 mg/kg/day, expressed as 42.1% mortality (8 of 17 pregnant animals), as well as an increase in early deliveries with three at this dose (and one each at all other doses). One doe at 2000 mg/kg/day aborted on GD 20 (no litters were aborted at any other doses). lncreases in early deliveries and abortion are usually considered indicative of maternal stress in rabbits. Pregnancy rate was high and equivalent across all groups from 0 - 1000 mg/kg/day (95.5, 95.7, 91.3, and 95.2%, respectively); pregnancy rate was slightly lower (8 1.8%) at 2000 mg/kg/day. Periodic maternal body weights and weight changes were statistically equivalent across all groups for all intervals evaluated. Maternal water consumption, expressed as g/animal/day or as g/kg/day was also statistically equivalent across all groups for all intervals, although water consumption appeared slightly increased for all EG-dosed groups during the treatment period but not in a manner related to dose. No clear treatment-related clinical signs of toxicity were observed. The 8 does which died at 2000 mg/kg/day died on GD 9 (one doe) and 11 (two does) and on GD 13, 14, 19, 21 and 25 (one doe each). At scheduled necropsy on GD 30 there were no significant effects of treatment on corrected (for uterine weight) maternal gestational weight change, gravid uterine weight, liver weight, or kidney weight at any dose. However, maternal absolute kidney weight (but not relative weight) was slightly increased at 2000 mg/kg/day to 106.3% of the control value for the right kidney (left kidney value was 107.6% of control). Histologic evaluation of maternal kidneys revealed treatment-related renal lesions only at 2000 mg/kg/day. The lesions were limited to the cortical renal tubules and included intraluminal crystals (appearance consistent with oxalate), epithelial necrosis, and tubular dilatation and degeneration. The most severe findings, crystals (designated "marked") and necrosis, were observed in the does which died on study, but the renal tubular necrosis observed in these animals was not a postmortem event. The cause of death in these animals was determined to be renal failure. There were no treatment-related effects on any gestational parameters, including no effects on number of ovarian corpora lutea, on total non-live or live implantation sites per litter, or on pre- or post-implantation loss. There was a statistically significant increase in the number of corpora lutea at 500 mg/kg/day associated, as expected, with a slight increase in the number of implantation sites/litter and a slight increase in live litter size. This finding was not observed at higher doses and is considered most likely due to biologic variation. The values at 500 mg/kg/day are still well within historical control values for these parameters.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in incidence of malformations pooled as external, visceral (including craniofacial), or skeletal, or as total malformations or variations. Examination of fetal malformations and variations by individual findings also indicated no findings which were treatment- or dose-related and none which appeared predominantly or exclusively at higher doses
Skeletal malformations:
not specified
Description (incidence and severity):
There were no treatment-related alterations in incidence of malformations pooled as external, visceral (including craniofacial), or skeletal, or as total malformations or variations. Examination of fetal malformations and variations by individual findings also indicated no findings which were treatment- or dose-related and none which appeared predominantly or exclusively at higher doses
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment-related alterations in incidence of malformations pooled as external, visceral (including craniofacial), or skeletal, or as total malformations or variations. Examination of fetal malformations and variations by individual findings also indicated no findings which were treatment- or dose-related and none which appeared predominantly or exclusively at higher doses
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no statistically significant or biologically relevant differences among groups for prenatal mortality, expressed as resorptions or dead fetuses, or for prenatal toxicity, expressed as fetal body weight/litter for all fetuses or for male and female fetuses separately. There were no treatment-related alterations in incidence of malformations pooled as external, visceral (including craniofacial), or skeletal, or as total malformations or variations. Examination of fetal malformations and variations by individual findings also indicated no findings which were treatment- or dose-related and none which appeared predominantly or exclusively at higher doses.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The sensitivity of NZW rabbits relative to that of Sprague-Dawley rats and Swiss mice for maternal and developmental toxicity from gavage administration of EG during organogenesis can be determined for maternal toxicity: rabbits > mice > rats, and for developmental toxicity: mice > rats > rabbits.