Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
The accompanying analytical sheet indicated a purity of 99.9%.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The 171 virgin male and 170 virgin female outbred albino rats referred to as the CD rats were received from Charles River Breeding Laboratories, Inc., Portage, MI, on June 12, 1990
- Age: males were 63 days old and females were 56 days old upon arrival.
- Weight at study initiation: males, approximately 250-300 g and females, approximately 175-200 g.
- Housing: in stainless steel wire-mesh cages (22 .5 cm x 15 .5 ca x 18 cm high)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22°C
- Humidity (%): 40-70%

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Timed-pregnant CDs dams were dosed daily with undiluted DEG or Milli-Q water on gd 6 through 15. All treatments were dosed by gavage using a 3 inch, 16 gauge, stainless steel dosing needle (Perfektume, Popper and Sons, Inc., New Hyde Park, NY) attached to a 2500 µL, 3.0 mL or 5.0 mL glass syringe (Becton-Dickinson), depending on the dose level. The dose volume was based on the dose group and the individual animals' most recent body weight (gd 6, 9 or 12). The doses employed were 0.0, 1.0, 4.0 or 8.0 ml/kg/day based on results from a dose range-finding study also performed on timed-pregnant CD rats. Control animals were dosed with deionized (Milli-Q) water at the top dose volume of 8.0 ml/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Since the test substance was administered undiluted, no homogeneity or dose level verification analyses were performed at BRRC. Poststudy characterization of the test substance indicated no significant conformational changes throughout the course of the study.
Details on mating procedure:
Rats were mated 1:1 (one male : one female) in stainless steel wire-mesh cages (22.5 cm x 31.0 cm x 18 .0 cm high) and the paperboard beneath the cages was checked daily for dropped copulation plugs (Hafer, 1970). Each male was used only once in this study . Successfully mated (plug-positive) females were housed singly in animal room 173 for the duration of the study. The day a dropped copulation plug was found was designated gestational day (gd) 0. Twenty-five (25) plug-positive females were assigned to each experimental group by a randomization procedure based on stratification of gd 0 body weights such that all groups were equivalent in both mean body weight and body weight range on gd 0. Mean body weights of females placed on study were 234 68 - 236.52 grams. The mating period for these animals was June 25-28, 1990 ; gd 0 was June 26-28, 1990.
Duration of treatment / exposure:
gestation day 6 - 15
Frequency of treatment:
once daily
Duration of test:
until gestation day 21
Doses / concentrationsopen allclose all
Dose / conc.:
1 other: mL/kg bw/day
Dose / conc.:
4 other: mL/kg bw/day
Dose / conc.:
8 other: mL/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
All females on study were weighed on gd 0, 6 (prior to onset of dosing), 9, 12, 15 (during the dosing period), 18 and 21. Food and water consumption were measured over three-day intervals throughout gestation (gd 0-21). All females were examined daily for clinical signs of toxicity.
All surviving study females were sacrificed on gd 21 by carbon dioxide asphyxiation. The sacrifice period was July 17 through July 19, 1990. The maternal body cavities were opened by midline thoracolaparotomy.
A gross necropsy was performed on all animals that died during the study. Pregnancy status was determined and maternal kidneys were fixed and examined microscopically.
Ovaries and uterine content:
The gravid uterus, ovaries (including corpora lutea), cervix, vagina and abdominal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. Maternal liver and uterine weights were determined.
Maternal kidneys (2) were also weighed, and then bisected (left longitudinally, right transversely) and fixed in buffered neutral 10% formalin. Maternal kidneys from all dams on study were examined microscopically. The uterus was externally examined for signs of hemorrhage, removed from the abdominal cavity and dissected longitudinally to expose the contents.
Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions (Salewski, 1964).


Fetal examinations:
All live and dead fetuses and resorption sites were noted and recorded.
All live fetuses were weighed and sexed. All fetuses were examined for external variations and malformations including cleft palate. All live fetuses in each litter were anesthetized by hypothermia and examined for thoracic and abdominal visceral abnormalities by modification of methods described by Staples (1974). One-half of the live fetuses (even-numbered fetuses from litters with an even number of live fetuses, odd-numbered fetuses from litters with an odd number of live fetuses) in each litter were decapitated and their heads were fixed in Bouin's solution for examination of craniofacial structures by sectioning methods modified from Wilson (1965; 1973) . All fetuses (50% intact, 50% decapitated) in each litter were eviscerated, fixed in ethanol, processed for skeletal staining with alizarin red S (Crary, 1962 ; Peltzer and Schardein, 1966), 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 (e.g. maternal body weights, organ weights, etc.) were intercompared for the three DEG-exposed groups and the 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.
Nonparametric data obtained following laparohysterectomy 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 probability value of p < of 0.05 (two-tailed) was used as the critical level of significance.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Cold extremities, slow or audible respiration and/or hypoactivity were observed in two out of the three females exposed to 8 mL/kg bw/day, before their death.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
- 3 of 25 females in the 8.0 mL/kg/day group died on gd 11 after six days of dosing.
- There were no treatment-related maternal deaths in the mid and low dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Maternal gestational body weights and weight gains were decreased in the 8.0 mL/kg bw/day group, especially during the first three days of treatment, gd 6-9.
- At scheduled sacrifice on gd 21, corrected gestational weight gain was slightly decreased in the 4.0 and 8.0 mL/kg bw/day groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was decreased in the 4.0 and 8.0 mL/kg bw/day groups on gd 6-9.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased during the treatment period in the 4.0 and 8.0 mL/kg bw/day 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 and liver weights were increased in the 8.0 mL/kg bw/day group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- All three dams that died (8 mL/kg bw/day dose group) showed evidence of moderate to severe microscopic kidney lesions consisting of tubular vacuolization and proteinosis.
- Upon microscopic evaluation of maternal kidneys, increased incidences of basophilia and interstitial nephritis were observed in dams of the 8.0 mL/kg bw/day group indicating repair of damaged renal tubulus.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

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:
no effects observed
Description (incidence and severity):
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:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related abortions, early deliveries or reductions in overall pregnancy rate. The number of females containing non-viable implants demonstrated no treatment-related associations. A total of 22-25 live litters were examined in each dose group.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
1 other: mL/kgbw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 8.0 mL/kg bw/day group, fetal body weights per litter (all fetuses, males or females) were significantly reduced.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
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
Description (incidence and severity):
There were no statistically significant increases in the incidences of malformations (individual, pooled external, visceral, skeletal or total) in any treatment group. There were no treatment-related differences among groups for individual external or visceral variations or for pooled external, visceral, skeletal or total variations.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant increases in the incidences of malformations (individual, pooled external, visceral, skeletal or total) in any treatment group. There were no treatment-related differences among groups for individual external or visceral variations or for pooled external, visceral, skeletal or total variations. Increased incidences of five individual skeletal variations indicative of delayed ossification were observed in the 8.0 mL/kg bw/day group. These included poorly ossified thoracic centrum # 10 and 13, thoracic centrum # m10 bilobed, poorly ossified interparietal, and anterior arch of the atlas split. Among those five variations, two, anterior arch of the atlas split and thoracic # centrum # 10 bilobed, exhibited an increased incidence in the 4.0 mL/kg bw/day group.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no statistically significant increases in the incidences of malformations (individual, pooled external, visceral, skeletal or total) in any treatment group. There were no treatment-related differences among groups for individual external or visceral variations or for pooled external, visceral, skeletal or total variations.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 other: mL/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
external: thorax
Description (incidence and severity):
Among those five variations, two, anterior arch of the atlas split and thoracic # centrum # 10 bilobed, exhibited an increased incidence in the 4.0 mL/kg bw/day group.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
4 other: mL/kg bw.day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, exposure of pregnant CD rats to diethylene glycol by gavage at 1.0, 4.0 and 8.0 mL/kg bw/day during organogenesis resulted in evidence of severe maternal toxicity, including death and kidney injury, at 8.0 mL/kg bw/day, and less severe maternal effects at 4.0 mL/kg bw/day. Developmental toxicity as evidenced by reduced fetal weight and delayed ossification was observed at 8.0 mL/kg bw/day. Minimal developmental toxicity was observed at 4.0 mL/kg bw/day. The "no observable effect level" (NOEL) for maternal toxicity was 1.0 mL/kg bw/day, and the NOEL for developmental toxicity was also 1.0 mL/kg bw/day. There was no evidence of teratogenic effects at any dosage.