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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The developmental toxicity/teratogenicity is read-across from the ethane-1,2-diol data. Rapid hydrolysis of the reaction mass to ethane-1,2-diol and formic acid has been shown (see toxicokinetics section) and justifies the read-across approach (see also justification atttached to section 13 of the IUCLID).
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessing the potential of ethane-1,2-diol, when administered by gavage during major organogenesis, to cause maternal and developmental toxicity in rabbits
GLP compliance:
not specified
Limit test:
no
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
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 bw 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 (GD) 6 to19
Frequency of treatment:
Daily
Duration of test:
Until gestation day (GD) 30
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
23 - 24 females/group
Control animals:
yes, concurrent no treatment
Details on study design:
The doses 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.
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 bw/day EG groups and all the kidneys from the 2000 mg/kg bw/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.
Clinical signs:
no effects observed
Description (incidence and severity):
Renal pathology but no other indicators of maternal toxicity
Mortality:
mortality observed, treatment-related
Description (incidence):
42.1% mortality (8 of 17 pregnant animals) at 2000 mg/kg bw/d
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).
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.
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
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, non-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:
no effects observed
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 bw/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:
no effects observed
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
One doe at 2000 mg/kg bw/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.
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:
no effects observed
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
increase in early deliveries with three at 2000 mg/kg bw/d (and one each at all other doses).
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Pregnancy rate was high and equivalent across all groups from 0 - 1000 mg/kg bw/day (95.5, 95.7, 91.3, and 95.2%, respectively); pregnancy rate was slightly lower (8 1.8%) at 2000 mg/kg bw/day.

Details on maternal toxic effects:
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.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
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.
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:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
No developmental effects were observed at 2000 mg/kg bw/day, the highest dose tested. The NOAEL for maternal toxicity was determined to be 1000 mg/kg bw/d.
Executive summary:

Rabbits were administered doses with 0, 100, 500, 1000 and 2000 mg/kg bw/d by gavage during gestation days 6 -19.

At 2000 mg/kg bw/d, severe maternal toxicity (mortality and degenerative changes in the kidney were observed but no developmental or reproductive effects in fetuses).

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The goal of the study was to establish no-observed-effect levels (NOELs) for maternal and developmental toxicity, including teratogenicity
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: 1 or 3 per cage, separated by sex
- Diet: ad libitum
- Water: the study females received drinking water in bottles with stainless steel sipper tubes to reduce stress and to allow water consumption measurements.- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24 °C
- Humidity (%): 50-58%
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Dosing solutions were prepared by adding the appropriate amount of test material in grams to a volumetric flask and diluting to volume with Millipore filtered water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
EG concentrations in dosing solutions were verified using a Hewlett-Packard 5880A gas chromatograph equipped with a flame ionization detector. EG concentrations in dosing solutions were 92.0 to 107.4% of nominal for mice. All dosing solutions were stable for at least 21 days when stored at room temperature.
Details on mating procedure:
The animals were bred 1:1. Each male was used only once in this study. 30 plug-positive females were randomly assigned to each of the experimental groups on gestation days (GD) 0 using a stratified randomization system. A total of 150 plug-positive females were used.
Duration of treatment / exposure:
gestation day (GD) 6 to 15
Frequency of treatment:
Daily
Duration of test:
until gestation day (GD) 18
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30 plug-positive females/dose
Control animals:
yes, concurrent no treatment
Maternal examinations:
All animals were observed daily (GD 0 - 18) for any clinical signs of toxicity and were weighed on GD 0, 6, 9, 12, 15 and 18. All suriving study females were sacrificed on GD 18. The maternal body cavities were opened by midline thoracolaporatomy. The gravid uterus, ovaries, cervix, vagina and abdominal and throacic cavities and organs were examined grossly. Ovarian corpora lutea of pregnancy were counted. Maternal liver, kidney and gravid uterus weights were determined. Maternal kidneys were bisected and retained in buffered neutral 10% formalin. Uteri were externally examined for signs of hemorrhage, removed from the peritoneal cavity and dissected longitudinally to expose their contents. Status of all implantation sites and resorption sites was noted and recorded. Uteri from females that appeared non-gravid were placed in a 10% ammonium sulfide solution for detection of early resorptions.

The fixed kidneys from dams surviving to scheduled sacrifice in the vehicle control and high dose group were embedded in paraffin, sectioned at 5 microns and stained with hematoxylin and eosin.
Ovaries and uterine content:
see "maternal examinations"
Fetal examinations:
All live fetuses were weighed, sexed and examined for external malformations including cleft palate and variations. All live fetuses were also examined for thoracic and abdominal visceral abnormalities by modification of methods and sex verified internally. All of the fetuses in each litter were eviscerated, fixed in ethanol, processed for skeletal staining and examined for skeletal malformations and variations.
Statistics:
The unit of comparison was the pregnant female or the litter. Results of the quantitative continuous variables were intercompared for the 4 test groups vs. the vehicle control group by use of the Levene's test for equal variances, analysis of variance (ANOVA) and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. Non-parametric data obtained following laparotomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test, when appropriate. Incidence data were compared using Fisher's Exact Test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical signs in surviving animals, which exhibited a dose-related incidence, or statistically significant increase.
The pregnant females which died or were sacrificed moribund across all groups (except at 150.0 mg/kg/kg) exhibited a number of clinical signs prior to death/sacrifice; none of the other females exhibited any clinical signs (except for one dam at 50.0 mg/kg/day which exhibited red urogenital discharge).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One to three females across all groups (except at 150.0 mg/kg/day) died or were sacrificed moribund. One (1) each at 0.0 and 50.0 mg/kg/day, three (3) at 500.0 mg/kg/day and two (2) at 1500.0 mg/kg/day died during the dosing period. Of these, one female each at 500.0 and 1500.0 mg/kg/day had as the proximate cause of death technical dosing error, but these animals were so physically compromised by previously observed clinical signs that the decision was made by the Study Director to retain their data and not remove them from study. The cause of the deaths appeared to be loss of blood from bleeding from the reproductive tract.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and weight gain were statistically equivalent across all groups at all time points and intervals measured. There were slight, but not statistically significant reductions in maternal weight for GD 12, 15 and 18, and similar reductions in maternal weight gain for GD 6 - 15 (treatment period) and for GD 0 - 18 (gestation period) at 1500.0 mg/kg/day.

Maternal terminal body weight and gestational weight change were slightly, but not significantly, reduced at 1500.0 mg/kg/day.
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
Description (incidence and severity):
Water consumption, expressed as g/animal/d exhibited no significant changes in any treatment group at any interval evaluated.
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:
no effects observed
Description (incidence and severity):
Liver and kidney weights were unaffected.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Maternal observations at scheduled necropsy did not differ significantly between the groups.

There was blood observed in the stomach, vagina, vulva, and/or urinary bladder in all of the animals except for the two animals (one each at 0.0 and 50.0 mg/kg/day), which were too autolyzed to confirm the findings. The two females whose proximate cause of death was technician error are documented as punctured oesophagus for the one at 500.0 mg/kg/day and as abscess in the subcutis and adhesion of the submandibular lymph node for the one at 1500.0 mg/kg/day. No parameters evaluated exhibited statistically significant differences in any treatment group relative to the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In kidneys from the top dose group there were no treatment-related histologic renal lesions and no oxalate crystals were observed in any of the kidney sections examined.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Dams with fully resorbed litters included none (0) at 0.0 mg/kg/day, one (1) each at 50.0 and 150 mg/kg/day, none (0) at 500.0 mg/kg/day and two (2) at 1500.0 mg/kg/day. A total of 19-24 litters were examined in each group.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The number of early resorptions and non-viable implants/litter were slightly increased at 1500 mg/kg/day, but these values were not statistically different from the values in the concurrent control group and are within the range of historical control values for these parameters in this species and strain.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Two (2) females at 0 mg/kg/day, three (3) at 50.0 mg/kg/day, two (2) at 150.0 mg/kg/day, one (1) at 500.0 mg/kg/day and one (1) at 1500.0 mg/kg/day delivered early.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate was approx. equivalent across all groups (84.0 -92.3%), except for the control group which had a 70.4% pregnancy rate (eight of 27 examined at scheduled sacrifice were not pregnant).
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related significant differences among groups in the number of corpora lutea/dam, the number of total, non-viable (early or late resorptions or dead fetuses) or viable implantations/litter, or on sex ratio (percentage male fetuses).

Key result
Dose descriptor:
NOEL
Effect level:
1 500 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights per litter were slightly reduced at 500 mg/kg bw/d
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There was no statistically significant increase in the incidence of any individual external or visceral malformations or of pooled external or visceral malformations.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The incidences of two individual malformations, of pooled skeletal malformations and of total malformations were all significantly increased at 500.0 mg/kg/day. The increased skeletal malformations were fusion of 2 -12 thoracic arches and fusion of 2 -12- ribs. In addition, a number of skeletal malformations occurred only at 1500.0 mg/kg/day, but the incidences were not statistically significantly different from those in the vehicle control group.

The incidence of 24 skeletal variations (out of 134 different findings observed) exhibited statistically significantly different values in one or more treatment groups relative to the vehicle control group. 23 of these were significant increases in incidence for the 1500.0 mg/kg/day group, including increased incidences of poorly ossified and unossified cervical centra (4 findings), poorly ossified and unossified thoracic centra (5 findings), extra thoracic centra and arch (1 finding), poorly ossified lumbar centra (4 findings), extra ribs (thoracic and lumbar, 2 findings), unossified bones of the forelimbs in the paws (proximal phalanges, 1 finding), poorly ossified sternebrae (3 findings), extra ossification site between sternebrae (1 finding), enlarged sagittal suturo (1 finding) and enlarged frontal fontanel (1 finding). In addition, 2 findings exhibited a significantly different incidence in groups other than the top dose group relative to those in the vehicle controls. There were an increased incidence of extra 14th rib on the first lumbar arch, bilateral, at 500.0 mg/kg/day (also observed at 1500.0 mg/kg bw/day), and a decreased incidence of poorly ossified frontal bone at 150.0 mg/kg/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
There was no statistically significant increase in the incidence of any individual external or visceral malformations or of pooled external or visceral malformations.
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant increases in any individual external or visceral variations, in variations by category (external, visceral or skeletal) or in total variations.

The incidence of pooled visceral and skeletal variations was 100% in all groups (one or more fetuses in all litters exhibited at least one finding, and in fact, all fetuses examined exhibited one or more skeletal variations) and the incidence of all variations were therefore also 100%.
Key result
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Under the conditions of this study, the NOEL for developmental toxicity in mice is at 150 mg/kg bw/d and the LOEL is 500 mg/kg bw/d
Executive summary:

Mice were dosed at 0, 50, 150, 500 or 1500 mg/kg bw/day by gavage during gestation days 6 -15.

There was no apparent treatment related maternal toxicity. Developmental effects observed at 1500 mg/kg/d included reduced body weights, fused ribs and arches, poor ossification in thoracic and lumbar centra, and increased occurrence of an extra 14th rib. At 500 mg/kg/d, slight reductions in fetal body weight and increased incidences of extra ribs were observed.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: EPA TSCA Testing Guidelines (1985; 1987)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: one group of 179 females was 56 days of age; The other group consisted of 32 females of 62 days of age
- Weight at study initiation: first group, approx. 175 - 210 g; second group, approx. 179 - 200 g
- Housing: in stainless steel wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
Rats were mated 1:1 (one male: one female) in stainless steel wire-mesh cages and the paperboard beneath the cages was checked daily for dropped copulation plugs. Each male was used only once in this study. Successfully mated (plug-positive) females were housed singly for the duration of the study. The day a copulation plug was found was designated gestational day (GD) 0.
Duration of treatment / exposure:
Gestation day 6-15
Frequency of treatment:
Daily
Duration of test:
Until gestation day (GD) 21
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 plug-positive females/dose
Control animals:
yes
Details on study design:
25 plug-positive females were assigned to each experimental group by a randomization procedure stratified by body weight such that all groups were equivalent in both mean body weight and body weight range on GD 0; mean body weights were 234.23 - 235.17 on GD 0. The dose volume was based on the dose group and the GD 0 body weight of each animal on study.
Maternal examinations:
All females on study were weighed on gestation day 0, 6 (prior to onset of dosing), 9, 12, 15 (during the dosing period), 18 and 21. All females were examined daily for clinical signs of toxicity. All surviving study females were sacrificed on gestation day 21.

Maternal liver, kidney and uterine weights were determined. Maternal kidneys were weighed, bisected (left longitudinally, right transversely) and fixed in buffered neutral 10% formalin for possible subsequent histopathological examination. Formalin-fixed kidneys from high dose and control animals were processed, embedded, sectioned and stained with haematoxylin and eosin, and the examined by light microscopy.
Ovaries and uterine content:
The maternal body cavities were opened by midline thoracolaparotomy. The gravid uterus, ovaries (including corpora lutea), cervix, vagina and abdominal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted.
The uterus was externally examined for signs of haemorrhage, removed from the abdominal cavity and dissected longitudinally to expose the controls. All live and dead fetuses and resorption sites were noted and recorded. Uteri from females appeared non-gravid were placed in a 10% ammonium sulfide solution for detection of early resorptions.
Fetal examinations:
All fetuses were examined for external malformations including cleft palate and variations. All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities. All live fetuses were weighed and sexed. One-half of the fetuses in each litter were decapitated and their heads were fixed in Bouin's solution for examination of craniofacial structures by sectioning methods. All fetuses in each litter were eviscerated, fixed in ethanol, processed for skeletal staining and examined for skeletal malformations and variations.
Statistics:
The unit of comparison was the pregnant female or the litter. Results of the quantitative variables were intercompared for the EG-exposed groups and the vehicle control group by use of Levene's test for equal variances, analysis of variance (ANOVA), and t-tests with Bonferroni probabilities for pairwise comparisons. When Levene's test indicated homogeneous variances and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test. Non-parametric data obtained following laparchysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test, when appropriate. Incidence data were compared using Fisher's Exact Test. For all statistical tests, a probability value of p < 0.05 (two tailed) was used as the critical level of significance.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weights prior to and during treatment were equivalent across dose groups. Subsequent to treatment, on GD 10 and 21, body weights were significantly reduced at 2500 mg/kg bw/day. Body weight gains were unaffected by test chemical administration at 150, 500 and 1000 mg/kg bw/day. At 2500 mg/kg bw/day, body weight gains were significantly reduced for the first 3 days of treatment (GD 6 - 9), for das 6 - 15 (the entire treatment period), subsequent to treatment (days 15 - 18 and 15 - 21) and for days 0 - 21 (the entire gestational period).

At sacrifice on GD 21, there were no significant differences in maternal terminal body weight or in corrected terminal body weight at 150, 500 or 1000 mg/kg bw/day. At 2500 mg/kg bw/day, terminal body weight was significantly reduced relative to control values. Corrected body weight change (gestational weight gain minus gravid uterine weight) was unaffected by test chemical treatment.
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):
Increased water consumption was observed at 2500 mg/kg bw/day for days 6 - 9, 9 - 12, 12 - 15 and for days 6 - 15 (the entire treatment period). Water consumption post-treatment was equivalent across dose groups.
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):
Kidney weights and relative kidney weight were significantly increased at 2500 mg/kg/day. At 1000 and 2500 mg/kg/day increased liver weights were observed.

At sacrifice on GD 21, there were no significant differences in gravid uterine weight at 150, 500 or 1000 mg/kg bw/day. At 2500 mg/kg bw/day, gravid uterine weight was significantly reduced relative to control values. Corrected body weight change (gestational weight gain minus gravid uterine weight) was unaffected by the treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related necropsy findings of the pregnant dams at scheduled sacrifice.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic evaluation of kidneys from dams at 2500 mg/kg/day indicated no treatment-related lesions.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Percentages of preimplantation and post-implantation losses were equivalent across groups.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rates ranged from 88.0 - 100% and were essentially equivalent for all dose groups.
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the number of ovarian corpora lutea, total, viable or non-viable (early and late resorptions and dead fetuses) implantations per litter, or on sex ratio.
Details on maternal toxic effects:
No females aborted or delivered early and no females were removed from study.
Key result
Dose descriptor:
NOAEL
Remarks:
Maternal toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
water consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights per litter were significantly reduced at 1000 and 2500 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The pooled incidences of external, soft tissue (visceral) and skeletal malformations and of total malformations were significantly increased at 2500 mg/kg/day. At 1000 mg/kg/day, increased incidence of skeletal malformations was observed. No dose-related increase in individual, pooled or total malformations were demonstrated in litters at 150 or 500 mg/kg/day. There were no treatment-related variations observed upon external examination of fetuses.
When the number of variations were combined by category (external, soft tissue or skeletal) or pooled entirely, no statistically significant increases in fetal variations were demonstrated.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Increased incidence of eight skeletal malformations, most involving the thoracic region, were observed at 2500 mg/kg/day. These included missing cervical arch 7, fused thoracic arches, missing thoracic centra and arches, missing thoracic arches, rudimentary ribs other than 13, missing ribs, fused ribs and short ribs other than 13. At 1000 mg/kg, two skeletal malformations, missing thoracic arches and missing ribs exhibited increased incidences.

Of 171 skeletal variations observed, 73 exhibited significantly increased incidences at 2500 mg/kg/day. Most (49 of 73; 67%) involved reduced ossification in the thoracic region. Increased incidences of 14 skeletal variants were also observed at 1000 mg/kg/day; these variations involved delayed ossification in the thoracic region.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
The incidence of gastroschisis, a defect in anterior abdominal wall closure which permits extrusion of the abdominal contents, was significantly increased at 2500 mg/kg/day. While there were no statically notable differences in the incidence of gastroschisis at 1000 mg/kg/day, the occurrence of incomplete closure of the body wall in another litter at 1000 mg/kg/day suggests a treatment-associated effect at the mid-high dose. Increased incidences of several visceral defects were observed at 2500 mg/kg/day. These included: hydrocephaly, lateral ventricle dilation with tissue depression, and umbilical hernia.

The incidence of a single visceral variation, fetal atelectasis, was increased at 2500 mg/kg/day. Two other visceral variations, dilated renal pelvis (unilateral) and dilated renal pelvis (bilateral) exhibited reduced incidences at 500 and 2500 mg/kg/day, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

The mean measured concentrations of the dosing formulations were 15.2 mg/mL for the 15 mg/mL (150 mg/kg bw/day solutions, 51.8 mg/mL for the 50 mg/mL (500 mg/kg bw/day) formulations, 107.8 mg/mL for the 100 mg/mL (1000 mg/kg bw/day) solutions and 255.4 mg/mL for the 250 mg/mL (2500 mg/kg bw/day) formulations. Homogeneity and stability analysis conducted on the 15 and 250 mg/mL solutions indicated that the formulations remained stable for at least 21 days when stored at room temperature.

Conclusions:
For maternal toxicity, the NOAEL was determined as 1000 mg/kg bw/day and for developmental toxicity as 500 mg/kg bw/day.

Executive summary:

Rats were exposed to 0, 50, 150, 500, 1000, or 2500 mg/kg bw/day during gestation days 6 -15.

There was no apparent treatment related clinical maternal observations. In the 2500 mg/kg bw/day group, dams had significantly decreased body weight, increased water consumption, decreased uterine weight, increased kidney weight, and increased relative liver weight. Developmental effects observed at 500 mg/kg bw/day included reduced body weights, extra or missing ribs, missing arches, and poor ossification in thoracic and lumbar centra. At 2500 mg/kg bw/day, in addition to skeletal malformations, there was gastroschisis, hydrocephaly, lateral ventricle dilated (tissue depressed), umbilical hernia, and atelectasis. 

Pregnant Sprague-Dawley rats to EG by gavage during organogenesis resulted in clear evidence of maternal toxicity at 2500 mg/kg bw/day and consistent evidence of fetotoxicity and teratogenicity at 1000 and 2500 mg/kg bw/day.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
The purpose of the study was to investigate the potential for developmental toxicity, including embryotoxicity, fetotoxicity and teratogenicity
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: three per cage, in stainless steel wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 69-72°F
- Humidity: 32-37%.
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Mass median aerodynamic diameter (MMAD):
2.3 µm
Details on exposure:
The test material was placed in one-gallon glass bottles and blanketed with nitrogen. The purity and stability were verified by analysis both before and after the study. No significant compositional changes occurred while the study progressed, except for a slight increase (0.2%) in water content. The test material remained nearly 100% pure throughout the study. The nitrogen and hydrogen were Ultra High Purity grade and the air was breathing quality. The inhalation chambers were constructed of stainless steel with glass windows for animal observation, with the dimensions 60 cm (height) x 101 cm (depth) x 101 cm (width). Chamber volume was approximately 900 litres and airflow ranged from 120 to 200 L/min, depending on desired exposure concentration. Chamber temperature and relative humidity were recorded at least five times per exposure with a minimum-maximum thermometer. Target concentrations were 0, 150, 1000, and 2500 mg/m3. These exposure concentrations were based on a range-finding study in pregnant rats and mice which employed concentrations of 0, 50, 250, 1250 and 2500 mg/m3. For the 2500 mg/m3 group, two generation systems, each with a 4-tube Laskin nebulizer, were used. For the 1000 mg/m3 group, a single generation system with a 4-tube Laskin nebulizer was used for Exposure Days 1 through 8. Beginning on Exposure Day 9 and going through Exposure Day 13, a 2-tube Laskin nebulizer was added to the generation system of the 1000 mg/m3 group in order to help achieve the desired concentration. For the 150 mg/m3 group, a 1-tube Laskin nebulizer was used for generation of the aerosol atmosphere. Compressed air supplied to each nebulizer created a negative pressure such that the ethylene glycol was aspirated into the tube where it was then dispersed as a fine liquid aerosol. The liquid aerosol was then introduced into the top of the exposure chamber where it was diluted to the desired concentration and dispersed through the chamber by filtered chamber supply air. The operating pressure of the nebulizers used to generate the 2500, 1000, and 150 mg/m3 target concentrations of ethylene glycol was approximately 30 psig. Chamber concentrations of ethylene glycol were analysed by both gravimetric determinations and impinger samples in order to determine the aerosol and total ethylene glycol concentrations, respectively. Each chamber atmosphere was analysed for ethylene glycol three times during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered, and the chamber airflow during the exposure period) were also calculated for each chamber. A Perkin-Elmer Model 3920B gas chromatograph (GC) equipped with a flame ionization detector was, used to monitor the ethylene glycol concentrations in the impinger solutions. Calibration of the gas chromatograph was done with liquid injections of standard solutions of ethylene glycol in distilled water prepared volumetrically. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were determined from log probability plots of the cumulative percentage mass collected on each impactor stage. The flow rate for all samples was 14.2 L/min. The sample collection times for the 2500, 1000, and 150 mg/m3 groups were generally 4, 10, and 120 minutes, respectively.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Mice were bred 1: 1 (one female 20-30 g: one male at least 25 g) and checked daily for vaginal copulation plugs. The date a copulation plug was found was designated GD 0. Twenty-five (25) plug-positive females were randomly assigned to each experimental group using a stratified randomization system on GD 0.
Duration of treatment / exposure:
Gestational days (GD) 6-15
Frequency of treatment:
6 hours/day
Duration of test:
Until GD 18
Dose / conc.:
0 mg/m³ air (analytical)
Remarks:
Corresponding to 0 mg/m3
Dose / conc.:
150 mg/m³ air (analytical)
Remarks:
Corresponding to 119 ± 13 mg/m3
Dose / conc.:
1 000 mg/m³ air (analytical)
Remarks:
Corresponding to 888 ± 149 mg/m3
Dose / conc.:
2 500 mg/m³ air (analytical)
Remarks:
Corresponding to 2090 ± 244 mg/m3
No. of animals per sex per dose:
25 plug positive females/dose
Control animals:
yes
Details on study design:
The exposure concentrations were based on a range-finding study in pregnant mice which employed concentrations of 0, 50, 250, 1250 and 2500 mg/m3.
Maternal examinations:
Clinical observations and maternal body weights were documented daily throughout gestation. At scheduled necropsy, maternal animals were evaluated for body weight, liver weight, kidney weight, gravid uterine weight.

Maternal body weights were taken on GD 0, 6, 9, 12, 15 and 18. At scheduled sacrifice on GD 18, mice were sacrificed by cervical dislocation.
Ovaries and uterine content:
At scheduled necropsy, maternal animals were evaluated for number of ovarian corpora lutea, and status of implantation sites, i.e., resorptions, dead fetuses, live fetuses.
Fetal examinations:
Fetuses were dissected from the uterus, counted, weighed, sexed, and examined for external, visceral, and skeletal malformations and variations.
Statistics:
The unit of comparison was the pregnant female or the litter.

Results of quantitative continuous variables were intercompared for the 3 exposure groups and the control group of each species by use of Levene's test for equal variances, analysis of variance and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test. Non-parametric data obtained following laparo-hysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test, when appropriate. Incidence data were compared using Fisher's Exact test. For all statistical tests, the fiducially limit of 0.05 (two-tailed) was used as the criterion for significance.
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs which appeared treatment-related included only wet fur for all ethylene glycol-exposed groups because of the aerosol.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All mouse dams survived to scheduled termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 and 2500 mg/m3, reduced body weight and weight gain during and after the exposure period.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 and 2500 mg/m3, reduced gravid uterine weight. No other organs were affected.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
One dam at 2500 mg/m3 was carrying a totally resorbed litter at termination. All other pregnant animals had one or more live foetuses at sacrifice.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The numbers of corpora lutes, total implantations and percentage preimplantation loss per dam were unaffected by treatment.
Key result
Dose descriptor:
NOAEC
Effect level:
150 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
total litter losses by resorption
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights per litter (male, female and total) were reduced at both 1000 and 2500 mg/m3.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of viable implantations per litter was reduced at 2500 mg/m3. The number of non-viable implantations per litter was elevated at both 1000 and 2500 mg/m3 because of a significant increase in late resorptions at 1000 mg/m3, and a significant increase in late resorptions and in dead fetuses at 2500 mg/m3. The number of early resorptions at 2500 mg/m3 was also elevated but not statistically.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex ratio was significantly reduced at 1000 mg/m3 but not at 2500 mg/m3, and appeared unrelated to treatment
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a significant increase in the incidence of a number of external malformations at 1000 and 2500 mg/m3, including cleft palate, exencephaly, blood in the amniotic sac (associated with exencephaly or meningocele), misshapen nasopharynx, protruding tongue, destruction of normal brain architecture, facial deformity.
The incidence of total external malformations was elevated at 1000 and 2500 mg/m3, as was the incidence of total malformations. Variations were also affected by exposure to ethane-1,2-diol, almost entirely at 1000 and 2500 mg/m3. There was a significant increase in the incidence of one external variation; flattened dorsal snout, at 2500 mg/m3.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a significant increase in the incidence of a number of skeletal malformations at 1000 and 2500 mg/m3, including fusion of vertebral arches (cervical, thoracic and lumbar), missing cervical arches, mal-aligned thoracic centra, ribs fused, forked, missing and extra (not the fourteenth which is a common variation but also the fifteenth), and abnormal facial bones: nasal, frontal, parietal and premaxillary, associated with facial deformity.

A large number of fetal skeletal variations exhibited treatment-related elevated incidences. These included the following at 2500 mg/m3: thoracic centra poorly ossified, split, mal aligned, or skewed, thoracic arches poorly ossified, extra ribs, wavy ribs, reduced caudal ossification, poorly ossified frontal, parietal, interparietal and hyoid bones, hole in squamosal, unossified proximal phalanges of the forelimb, and poorly ossified or unossified metatarsals of the hindlimb. At 1000 and 2500 mg/m3, the following exhibited elevated incidences relative to those of controls: poorly ossified or unossified cervical centra, poorly ossified anterior arch of the atlas, unossified or bilobed thoracic centra, extra thoracic centra and arches, poorly ossified, bilobed or split lumbar centra, curved ribs, extra (fourteenth ) ribs, short ribs (other than the thirteenth), poorly ossified nasal bones, zygomatic arch, mandible, and squamosal, posteriorly enlarged sagittal suture line, enlarged fontanels, poorly ossified or unossified proximal phalanges of the forelimbs and hindlimbs, unossified intermediate phalanges of the forelimb and hindlimb, poorly ossified metacarpals of the forelimb, and unossified, split, poorly ossified or fused sternebrae.
Those skeletal variations which exhibited elevated incidences at all three ethylene glycol concentrations relative to controls included only 1-6 thoracic centra poorly ossified, and poorly ossified premaxillary. Other findings exhibited incidences significantly different from those of controls but these did not exhibit a dose-response and/or were of reduced frequency relative to that of controls.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There was a significant increase in the incidence of a number of visceral malformations at 1000 and 2500 mg/m3.

Visceral variations with an elevated incidence included distorted pattern of rugae on palate, pale spleen, dilated third ventricle of the diencephalon, and displaced cochlea at 2500 mg/m3, short nasal passages at 1000 and 2500 mg/m3, and dilated lateral ventricles of the cerebrum with and without brain tissue depression at all three ethylene glycol concentrations. Partial fetal atelectasis was reduced at 1000 mg/m3 relative to controls.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
150 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: cranium
external: face
skeletal: skull
skeletal: rib
skeletal: vertebra
visceral/soft tissue: central nervous system
visceral/soft tissue: ear
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/m³ air (analytical)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Executive summary:

Following exposure of mice to ethane-1,2 -diol aerosol during organogenesis, evidence of maternal and embryo fetal toxicity, including teratogenicity, was observed at 1000 and 2500 mg/m³. There were no observable effects to the mouse dams or conceptuses at 150 mg/m³. The amount of EG on the fur, groomed and ingested by the mice, may have been sufficient, per se, to produce the teratogenicity observed in mice in the present study. The role of inhaled ethylene glycol on teratogenesis cannot be determined from the present study.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Principles of method if other than guideline:
The purpose of the study was to investigate the potential for developmental toxicity, including embryotoxicity, fetotoxicity and teratogenicity
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: CD
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: in stainless steel wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 69-72°F
- Humidity: 32-37%.
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Mass median aerodynamic diameter (MMAD):
2.3 µm
Details on exposure:
The test material was placed in one-gallon glass bottles and blanketed with nitrogen. The purity and stability were verified by analysis both before and after the study. No significant compositional changes occurred while the study progressed, except for a slight increase (0.2%) in water content. The test material remained nearly 100% pure throughout the study. The nitrogen and hydrogen were Ultra High Purity grade and the air was breathing quality. The inhalation chambers were constructed of stainless steel with glass windows for animal observation, with the dimensions 60 cm (height) x 101 cm (depth) x 101 cm (width). Chamber volume was approximately 900 litres and airflow ranged from 120 to 200 L/min, depending on desired exposure concentration. Chamber temperature and relative humidity were recorded at least five times per exposure with a minimum-maximum thermometer. Target concentrations were 0, 150, 1000, and 2500 mg/m3. These exposure concentrations were based on a range-finding study in pregnant rats and mice which employed concentrations of 0, 50, 250, 1250 and 2500 mg/m3. For the 2500 mg/m3 group, two generation systems, each with a 4-tube Laskin nebulizer, were used. For the 1000 mg/m3 group, a single generation system with a 4-tube Laskin nebulizer was used for Exposure Days 1 through 8. Beginning on Exposure Day 9 and going through Exposure Day 13, a 2-tube Laskin nebulizer was added to the generation system of the 1000 mg/m3 group in order to help achieve the desired concentration. For the 150 mg/m3 group, a 1-tube Laskin nebulizer was used for generation of the aerosol atmosphere. Compressed air supplied to each nebulizer created a negative pressure such that the ethylene glycol was aspirated into the tube where it was then dispersed as a fine liquid aerosol. The liquid aerosol was then introduced into the top of the exposure chamber where it was diluted to the desired concentration and dispersed through the chamber by filtered chamber supply air. The operating pressure of the nebulizers used to generate the 2500, 1000, and 150 mg/m3 target concentrations of ethylene glycol was approximately 30 psig. Chamber concentrations of ethylene glycol were analysed by both gravimetric determinations and impinger samples in order to determine the aerosol and total ethylene glycol concentrations, respectively. Each chamber atmosphere was analysed for ethylene glycol three times during each 6-hour exposure. Daily nominal concentrations (an estimated concentration calculated from the amount of test material delivered, and the chamber airflow during the exposure period) were also calculated for each chamber. A Perkin-Elmer Model 3920B gas chromatograph (GC) equipped with a flame ionization detector was, used to monitor the ethylene glycol concentrations in the impinger solutions. Calibration of the gas chromatograph was done with liquid injections of standard solutions of ethylene glycol in distilled water prepared volumetrically. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were determined from log probability plots of the cumulative percentage mass collected on each impactor stage. The flow rate for all samples was 14.2 L/min. The sample collection times for the 2500, 1000, and 150 mg/m3 groups were generally 4, 10, and 120 minutes, respectively.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Rats were mated 1:1 (one male at least 250 g: one female 200-300 g) in stainless steel wire-mesh cages and the paperboard beneath the cages was checked twice daily for dropped copulation plugs. Successfully mated (plug-positive) females were housed singly in stainless steel wire-mesh cages for the duration of the study except during daily exposures. The day a copulation plug was found was designated gestational day (GD) 0.

Twenty-five (25) plug-positive females were randomly assigned to each experimental group using a stratified randomization system on GD 0. For exposures, plug-positive females were transferred, one per cage, to stainless steel wire-mesh exposure cages and the cage carriers, with stainless steel shelves beneath each row of cages, were moved into the chambers. Food and water were withheld-during exposures.

Duration of treatment / exposure:
Gestational Day (GD) 6-15
Frequency of treatment:
6 hours/day
Duration of test:
Until GD 21
Dose / conc.:
0 mg/m³ air (analytical)
Remarks:
Corresponding to 0 mg/m3
Dose / conc.:
150 mg/m³ air (analytical)
Remarks:
Corresponding to 119 ± 13 mg/m3
Dose / conc.:
1 000 mg/m³ air (analytical)
Remarks:
Corresponding to 888 ± 149 mg/m3
Dose / conc.:
2 500 mg/m³ air (analytical)
Remarks:
Corresponding to 2090 ± 244 mg/m3
No. of animals per sex per dose:
25 plug positive females/dose
Control animals:
yes
Details on study design:
The exposure concentrations were based on a range-finding study in pregnant rats which employed concentrations of 0, 50, 250, 1250 and 2500 mg/m3.
Maternal examinations:
Clinical observations and maternal body weights were documented throughout gestation. Maternal food and water consumption was measured in rats only throughout gestation. At scheduled necropsy, maternal animals were evaluated for body weight, liver weight, kidney weight, gravid uterine weight.

Maternal body weights were taken on GD 0, 6, 9, 12, 15, 18, and 21. At scheduled sacrifice on GD 21, rats were sacrificed by carbon dioxide asphyxiation.
Ovaries and uterine content:
At scheduled necropsy, maternal animals were evaluated for number of ovarian corpora lutea, and status of implantation sites, i.e., resorptions, dead fetuses, live fetuses.
Fetal examinations:
Fetuses were dissected from the uterus, counted, weighed, sexed, and examined for external, visceral, and skeletal malformations and variations.
Statistics:
The unit of comparison was the pregnant female or the litter.

Results of quantitative continuous variables were intercompared for the 3 exposure groups and the control group of each species by use of Levene's test for equal variances, analysis of variance and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t-test.
Non-parametric data obtained following laparo-hysterectomy were statistically treated using the Kruskal-Wallis test followed by the Mann-Whitney U test, when appropriate. Incidence data were compared using Fisher's Exact test. For all statistical tests, the fiducially limit of 0.05 (two-tailed) was used as the criterion for significance.
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs, which increased in incidence with increasing exposure concentrations, included only red fur discoloration on the head and neck, a nonspecific indication of stress, observed in all groups.
Mortality:
no mortality observed
Description (incidence):
All rat dams survived to scheduled termination.
Body weight and weight changes:
no effects observed
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
Ophthalmological 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):
Minimal maternal toxicity was indicated by a significant increase in absolute and relative liver weight at 2500 mg/m3. All other organs were not affected.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
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:
no effects observed
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
1 000 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was some evidence of treatment-related reductions in ossification of the fetal skeleton. These included an increase in the incidence of poorly ossified humerus (upper arm) and zygomatic arch (face) at 2500 mg/m3 and an increase in the incidence of poorly ossified metatarsals and proximal phalanges of the hindlimb at 1000 mg/m3 (but not at 2500 mg/m3). One additional incidental finding, that of bilobed sternebra number 1, exhibited a decreased incidence at 2500 mg/m3 relative to that of controls.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of total visceral malformations was significantly lower at 150 and 2500 mg/m3 relative to controls because of the finding of hydroureter and hydronephrosis in control litters, a malformation to which the CD rat is predisposed.
Key result
Dose descriptor:
NOAEC
Effect level:
150 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: NOAEC from inhalation exposure alone cannot be determined due to confounding oral exposure during whole-body exposure. There was no maternal or embryofetal toxicity at 150 mg/m3 and no teratogenicity at any aerosol concentration employed.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Exposure of rats during organogenesis resulted in minimal maternal toxicity at 2500 mg/m3 and minimal fetotoxicity at 1000 and 2500 mg/m3. There was no maternal or embryofetal toxicity at 150 mg/m3 (the no observable effect level) and no teratogenicity at any aerosol concentration employed.

Data source

Materials and methods

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

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
mortality

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion