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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Comparable to Guideline study. Well conducted and reported study.

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Test substance: 5-Ethylidene-2-norbornene (CAS No.16219-75-3)
- Source: Union Carbide Corporation, South Charleston, West Virginia, Lot No. 1609
- Analytical Purity: 99%

Test animals

CD (Sprague-Dawley)
Details on test animals or test system and environmental conditions:
Each animal was assigned a unique number and was ear tagged for identification.

- Virgin male and female CD (Sprague Dawley).
- Age not recorded; acclimated for 2 weeks before exposure commenced.
- Weight at study initiation: females ~235 g.
- Housing: 2 (1M/!F) to a cage in stainless steel wire-mesh cages.
- Diet: Prolab Certified Rodent Food@, ad libitum except during exposure period
- Water: tapwater
- Acclimation period: two weeks during which time they were examined by the Clinical Veterinarian and representative animals were subjected to a full necropsy, examination for intestinal parasites, histologic examination of selected tissues and serum viral antibody analysis.

- Temperature (exposure chamber): 20.4-22.2 Celsius
- Humidity (exposure chamber): 47.5-49.6%
- Photoperiod: 12-hour

Administration / exposure

Route of administration:
Type of inhalation exposure (if applicable):
whole body
unchanged (no vehicle)
Details on exposure:
For generation of atmospheres, liquid ENB was metered from a heated glass evaporator similar in design to that described by Carpenter et al. (1975) and, more recently, Snellings and Dodd (1990). The temperature in the evaporator(s) was maintained at the lowest level sufficient to vaporize the liquid (31-52°C). The resultant vapor was carried into the chamber by passage of conditioned air through the evaporator(s). Concentrations of the ENB vapor in the exposure chambers were monitored using a gas chromatograph equipped with a flame ionization detector.

Chamber volume: 3220litres, airflow 1000litres/min.

Chamber temperature, relative humidity, and airflow rate were recorded approximately every 30 minutes during each six-hour exposure.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytical chamber concentrations were measured by gas chromatography. Calibration of the gas chromatograph was done using a series of standards which encompassed the entire range of vapor concentrations generated in the exposure chambers. Each chamber atmosphere was monitored for concentration verification approximately once every 25 minutes during each daily six-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 calculated for each chamber. In a preliminary study on the distribution of vapur in the exposure chamber, each chamber was sampled at 5 different positions in triplicate. The variation seen was deemed acceptable.
Details on mating procedure:
- If cohoused:
- M/F ratio per cage: 1:1 (one male at least 300 g:one female at least 200 g) mating cages (22.5 x 31.0 x IS cm high)
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Plug-positive females-were housed individually for the duration of the study. 
Duration of treatment / exposure:
Gestation days (GD) 6-15
Frequency of treatment:
6 hours/day
Doses / concentrationsopen allclose all
Dose / conc.:
123 mg/m³ air (analytical)
Dose / conc.:
492 mg/m³ air (analytical)
Dose / conc.:
1 740 mg/m³ air (analytical)
No. of animals per sex per dose:
Control animals:
other: yes, air-exposed only
Details on study design:
Sex: female
Duration of test: females sacrificed on GD21


Maternal examinations:
- Time schedule: Maternal animals were observed daily for mortality, morbidity, and signs of toxicity

- Time schedule for examinations: Body weights were recorded on GD 0, 6, 9, 12, 15, 18, and 21.

FOOD CONSUMPTION : Yes, Maternal food consumption was measured at 3-day intervals.

- Sacrifice on gestation day # 21
- Organs examined: general abdominopelvic gross pathology, gravid uterine weight, liver and kidney weights. Maternal thyroid glands were weighed and prepared for light microscopic examination and morphometric measurements.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: not reported
Fetal examinations:
All fetuses were weighed, sexed, and examined for external variations and malformations.
- Soft tissue examinations: Yes: half per litter were examined for thoracic and abdominal abnormalities ; these fetuses were decapitated and the heads fixed in Bouin's solution for examination of craniofacial structures by sectioning. 
- Skeletal examinations: Yes: All fetuses were eviscerated and processed for skeletal staining with alizarin red S to examine for skeletal malformations and variations.
- Head examinations: Yes: half per litter
Quantitative continuous variables were intercompared between ENB groups and the corresponding air-control groups using Levene's test for equal variances,analysis of variance, and t-tests. The t-test was used when the F value from the ANOVA was significant. Non-parametric data were statistically analyzed using the Kruskall-Wallis test followed by the Mann-Whitney U test. Incidence data were compared using Fisher's Exact Test. A probability value 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:
no effects observed
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Gestational body weights were reduced GD 6-9, 9-12, 12-15, and after exoosure ceased GD 15-18 in the high dose animals and GD 12-15 in the mid dose animals. Gestational body weight gains were statistically significantly reduced (p< 0.01) 61.5% over GD 6-15 in the high dose animals and 25.2% over GD 9-15 in the mid dose animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantly reduced overall during the exposure period in both the mid and high dose groups. For the mid dose group the main reduction occured between days 9 and 15 whereas in the high dose group the reduction was seen during the whole exposure period (GD 6-15). Food consumption was similar to controls in the post exposure period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effect on blood T3 and T4 hormone levels (free and total)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute liver weight slightly increased in high dose animals and relative weight signficantly increased in mid and high dose animals. No effect on thyroid weight.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Light microscopy of thyroid from all groups revealed colloid depletion characterised b small, collapsed or angular shaped follicles containing minimal colloid. Both the numbers of animals showing depletion and the degree of depletion were greater in the ENB exposed animals and showed a dose response. Morphometric quantification of follicular colloid revealed an exposure-concentration related depletion of colloid. There were no clinical signs suggestive of thyroid dysfunction

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:
effects observed, non-treatment-related
Description (incidence and severity):
One dam at 100 ppm delivered early. This was not regarded as significant.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rates ranged from 83.3 to 100% and were equivalent across all groups. Between 20 and 25 litters were available for examination in each group.
Other effects:
no effects observed
Description (incidence and severity):
Gravid uterine weight unaffected

Effect levels (maternal animals)

Dose descriptor:
Effect level:
123 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Maternal abnormalities

no effects observed

Results (fetuses)

Fetal body weight changes:
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:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Three skeletal variants were increased in litters of the high dose group (bilobed 12th thoracic centrum, split 12th thoracic centrum, and poorly ossified second sternabra) and one variant (bilobed 12th thoracic centrum) was increased in the mid dose group litters, but these occurred in the presence of maternal toxicity and were not seen in the 25 ppm group. Whilst the incidence of bilobed 12th thoracic centrum was statistically increased relative to controls, there was no clear dose response relationship - incidence at all three doses was similar and the effect did occur in controls 6% in controls versus 16%, 21% and 21% in the low, medium and high dose group animals. The biological significance is therefore questionable in the absence of any other significant findings and this finding was considered non-adverse
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:

Effect levels (fetuses)

Dose descriptor:
Effect level:
492 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: skeletal variations

Fetal abnormalities

no effects observed
Description (incidence and severity):
Excludes minor skeletal variations

Overall developmental toxicity

Developmental effects observed:

Applicant's summary and conclusion

Executive summary:

Neeper-Bradley et al. (1995) reported results of a teratogenicity test of ENB conducted in CD (Sprague-Dawley) rats by inhalation of 0, 25, 100 and 354 ppm (0, 123, 492, 1740 mg/m3) during days 6-15 of pregnancy. There was no maternal mortality. Maternal body weights, body weight gain, and food consumption were reduced over the exposure period at 100 and 354 ppm, with partial or complete recovery post exposure. Increased relative liver weights were measured for the 100 and 354 ppm groups. Vacuolar depletion of thyroid follicular colloid was present in all groups of pregnant animals, including air-only controls, but the incidence and extent was greater in ENB vapor-exposed rats. There were no effects on tri-iodothyronine (T3), tetra-iodothyronine (T4) or T3 uptake. There were no increases in the incidence of malformations or external and visceral variations. Three skeletal variants (bilobed 12th thoracic centrum, split 12th thoracic centrum, and poorly ossified second sternabra) were increased at 354 ppm, and one (bilobed 12th thoracic centrum) was increased at 100 ppm. Thus, minimal developmental toxicity in the form of skeletal variants was observed in the 100 and 354 ppm group litters in the presence of maternal toxicity but analysis of the results suggests no dose response relationship and, in the absence of other findings, this change may not be biologically significant. On this basis, it is concluded that the NOAEL for maternal toxicity is 25ppm (123 mg/m3) whilst that for developmental toxicity/teratogenicity is 100 ppm (492 mg/m3)