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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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
; only a single dose was tested.
Principles of method if other than guideline:
The aim of the study was to evaluate the embryo/fetal toxicity and teratogenic effects of the test substance when administered by gavage to pregnant rats from day 6 to day 15 of gestation. The test material was administered to a group of 22 female rats at a single dose level of 750 mg/kg bw. Another group served as a common control and received only corn oil.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,6-Hexanediol diacrylate; coded as C-255
- Analytical purity: no data given
- Impurities (identity and concentrations): no data given
- Lot/batch No.: 3-81
- Physical state: light amber liquid
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Kingston, New York
- Age at study initiation: ca. 5 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: individual in elevated wire-mesh cages; during mating two females were housed with one male
- Diet: Purina Rodent Laboratory Chow, ad libitum
- Water: ad libitum
- Acclimation period: ca. 9 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24-25 °C
- Humidity (%): 57 +- 4.8 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The amount of compound required for each group was weighed, filled to volume with the vehicle and stirred on a magnetic stirrer. The prepared dilutions were transferred to amber bottles and labelled. Before use, the prepared dilutions were well shaken and the animals were dosed while the solutions were mixed on a magnetic stirrer. Fresh solutions were prepared weekly.

VEHICLE:
- Duke´s Corn Oil, a yellow liquid was received from the C.F. Sauer Co. Richmond, Virginia and used as vehicle.
- Lot/batch no.: 80235
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Following a health status examination by a staff veterinarian, the males and females (1 male per 2 females) were placed in breeding cages for a maximum of 3 weeks. Females were rotated after the tenth day of mating. Mating was confirmed by the presence of a vaginal plug or by daily examination of vaginal smears for the presence of sperm. The day that mating was confirmed was designated as day 0 of gestation for each female placed on study.
Duration of treatment / exposure:
day 6 to day 15 of gestation
Frequency of treatment:
daily
Duration of test:
until day 20 of gestation
Doses / concentrations
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a range-finding study.
Test compound (HDDA) was evaluated for tolerance in pregnant rats to determine dose levels for a subsequent teratology screening study. Test substance was administered by gavage at 100, 500 and 1000 mg/kg/day (6 inseminated females per dose) from Days 6-15 of gestation. At the dose of 1000 mg/kg/day, clinical signs, decrease of bodyweight were observed. As the dose of 100 and 500 mg/kg/day were well tolerated by females, the dose of 750 mg/kg/d was selected to evaluate the embryo/fetal toxicity and teratogenic effects of HDDA.

Examinations

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: mortality, moribundity and clinical signs
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0, 6, 9, 12, 15 and 20 of gestation

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: on day 6-8, 9-11, 12-14, 15-17 and 18-20 of gestation

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 20:
On day 20 of gestation, females were sacrificed by carbon dioxide asphyxiation and the fetuses were taken by cesarean section. Following gross examination of each dam, the number of corpora lutea per ovary and the number and placement of implantation sites, early and late resorptions, and live and dead fetuses in each uterine horn were recorded. Fetuses were removed from the placenta, individually identified, examined externally, weighed, sexed, and measured from the frontal-parietal suture to the base of the tail (crown-rump distance). Gravid and nongravid uterine weights (with ovaries attached) were recorded.
- Organ Weights:
Following careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner, the gravid and nongravid uterine weights (with ovaries attached) of each female were taken for organ weighing.
- Tissue Preservation:
The uterus and ovaries of each dam, in addition to unusual lesions, were preserved in 10 % neutral buffered formalin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- Visceral Examination of Fetuses:
Approximately one-third of the fetuses from each litter were fixed in Bouin's solution, sectioned, and examined by Wilson's freehand razor technique (Wilson and Warkany, 1965), and sealed in plastic. The prepared sections were re-examined against a light box with the aid of magnification.
- Skeletal Examination of Fetuses:
After undergoing external examinations, approximately two-thirds of all fetuses from each litter were opened by longitudinal incision and the viscera examined grossly. The fetuses were then placed in M5 ethyl alcohol and the skeletons were stained in a potassium hydroxide - Alizarin red S solution (modified Staples and Schnell, 1964). Each skeleton was examined with the aid of magnification on a light box for bone alignment, degree of ossification, and anomalies. The number of sternebrae, ribs, caudal vertebrae, and bones of the extremities were noted and recorded. The fetuses examined by Wilson's technique were preserved In Bouin's solution and sealed in plastic after sectioning. The fetuses stained for skeletal examination were preserved in plastic in a glycerin ethanol (1:1) solution with several crystals of thymol to retard bacterial growth.

Statistics:
Survival was statistically analyzed by the National Cancer Institute Package (Thomas, Breslow, & Gart, 1977).
The mean maternal body weight changes (Days 0-6, 6-15, 15-20, and 0-20), mean maternal food and water consumptions (Days 6-9, 9-12, 12-15, 15-18, 18-20, and 6-20), percent males per litter, mean fetal body weights and lengths, fetal viability, percent resorptions, implantation efficiency, gravid and nongravid uterine weights, and the incidence of visceral and skeletal anomalies and variants were analyzed by Box's test for homogeneity of variances (Box, 1949). This test was followed by a one-way classification analysis of variance (ANOVA), if the variances proved to be homogenous. If the variances proved to be heterogenous, a rank transformation of data was performed, which was followed by Box´s test and ANOVA. If ANOVA of untransformed or transformed data was significant, Dunnet´s T-test was used for control vs. treatment group mean comparisons.
Pregnancy rates were analyzed by a test of multiple proportions using one degree of freedom Chi-square test with Yates´continuity correction. All pairwise comparisons were evaluated at the 5.0 % probability (one-tailed) level.
Indices:
no
Historical control data:
no

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
An increased incidence of clinical symptoms was noted (wheezing, dyspnea, urine stains, wasted feed, rough haircoat, hunched pasture, bloody crust on the eyes, nase, snouth, and frontpaws, salivation and alopecia).
Mortality:
no mortality observed
Description (incidence):
No mortality occured during treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weight changes lower than the control, but not statistically significant were observed in the treated group during the treatment phase.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the treated group a statistically significant increase in water consumption was noted on days 18 and 20 and in total water consumption, compared to control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The most distinct alterations attributed to treatment were noted in the stomachs of the treated animals. The findings consisted of discoloured material or fluid in the stomach; gas distending the stomach; discoloured, ulcerated or raised areas in the glandular or nonglandular portion of the stomach. glandular mucosa smooth; walls thick or thin/smooth; and nonglandular mucosa thin and pale, or thick and rough. Other gross pathology findings were considered to be incidental in nature and showed no relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Dead fetuses:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Results were comparable between treated and controls animals.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Results were comparable between treated and controls animals.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
LOAEL
Remarks:
(maternal systemic toxicity)
Effect level:
750 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
effects observed, treatment-related

Results (fetuses)

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:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal examinations included incidences of cleft palate in 1 pup. A high incidence of dilated ureters was noted in both treated and control groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
A higher than control mean incidence of visceral variants was noted (but not statistically significant). Statistically significant increased mean incidence of skeletal variants was observed. The majority of the findings were due to delayed ossification of the various bone structures examined. The authors suggested that this response was resulting from the maternal toxicity noted.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A higher than control mean incidence of visceral variants was noted (but not statistically significant).
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Remarks:
(developmental toxicity)
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
effects observed, treatment-related

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Maternal toxicity and foetoxicity on rats were observed in this developmental study at the dose of 750 mg/kg/d. The embryotoxic effects resulted probably from maternal toxicity.
Executive summary:

Test compound (HDDA) was evaluated for tolerance in pregnant rats to determine dose levels for a subsequent teratology screening study. Test substance was administered by gavage at 100, 500 and 1000 mg/kg/day (6 inseminated females per dose) from Days 6-15 of gestation. At the dose of 1000 mg/kg/day, clinical signs, decrease of bodyweight were observed. As the dose of 100 and 500 mg/kg/day were well tolerated by females, the dose of 750 mg/kg/d was selected to evaluate the embryo/fetal toxicity and teratogenic effects of HDDA.

In the main study, the test substance was administered by gavage from Days 6-15 of gestation to 22 females at the dose of 750 mg/kg/d.

Compound-related maternal toxicity was observed: increased incidence of clinical signs, slight decrease of body weight gains and an increased incidence of gross pathology findings (stomach).

Pregnancy rates, corpora lutea and implantations, and mean implantation efficiency (number of implantations per number of corpora lutea) were generally comparable for treated animals and controls.

A higher than control number of fetuses exhibiting skeletal variants was observed in the HDDA group. The incidence was found to be significant both skeletal and lagging ossification. Taken together, maternal toxic effects as well as embryotoxic effects were observed. According to the authors, the embryotoxic effects resulted from maternal toxicity.