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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:
supporting study
Study period:
25 Mar - 19 Apr 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Guidelines for the Testing of Chemicals Section 4: Health Effects (Ministry of Environmental Protection of People's Republic of China)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
There are major deviations from the current OECD guideline. For details, please refer to "Principles of method if other than guideline".
Principles of method if other than guideline:
Deviations from the current OECD 414 guideline included the following:
dams were exposed on gestation day 6-15, the thyroid gland was not weighed or histopathologically assessed, no thyroid hormone levels were measured, 1/3 fetuses from each litter were examined for soft tissue alterations and all fetuses per litter were examined for skeletal alterations, anogenital distance was not measured, no individual data or raw data was included in the study report, the developmental effects were not specified as variations and malformations and the study was not conducted according to GLP.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
482-140-6
EC Name:
-
Cas Number:
13641-96-8
Molecular formula:
Hill formula: C6 H7 N O3 CAS formula: C6 H7 N O3
IUPAC Name:
2-isocyanatoethyl prop-2-enoate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Shanghai SLAC Laboratory Animal Co., Ltd., China
- Age at study initiation: 3 months
- Weight at study initiation: 269 - 339 g
- Housing: Animals were housed in animal barrier system.
- Diet: standard diet (Suzhou Shuangshi Laboratory Animal Feed Co., Ltd.), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70

IN-LIFE DATES: From: 25 Mar 2010 To: 19 Apr 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was mixed with corn oil. Animals were gavaged based on 10 mL/kg bw.

VEHICLE
- Justification for use and choice of vehicle: not provided
- Amount of vehicle (if gavage): 10 mg/kg bw
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: not provided
- Length of cohabitation: not provided
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: vaginal plug; referred to as Day 0 of pregnancy
Duration of treatment / exposure:
Pregnant rats were administered the test substance by oral gavage from Day 6 to 15.
Frequency of treatment:
once daily
Duration of test:
until Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0.88 mg/kg bw/day (actual dose received)
Dose / conc.:
4.4 mg/kg bw/day (actual dose received)
Dose / conc.:
22 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 pregnant female rats in the vehicle control group and 23 pregnant female rats per dose in the groups treated with the test substance.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on the results of an acute oral toxicity study. Groups of 5 female rats were orally dosed with 21.5, 46.4, 100, 215 and 464 mg/kg bw. The test substance was dissolved in corn oil and applied with a dose volume of 10 mL/kg bw. Mortalities were observed as follows:
21.5 mg/kg bw: No deaths occurred.
46.4 mg/kg bw: 1/5 females
100 mg/kg bw: 2/5 females
215 mg/kg bw: 4/5 females
464 mg/kg bw: 5/5 females

Observed clinical signs were as follows:
21.5 mg/kg bw: No obvious clinical signs were observed.
46.4 mg/kg bw: Salivation, unresponsiveness, gait flabby, sternutation, and emiction incontinence were observed at the second day after dosing in 1/5 females.
100 mg/kg bw: Salivation, unresponsiveness, gait flabby, sternutation, and emiction incontinence were observed 10 min after dosing in 2/5 females.
215 mg/kg bw: Salivation, unresponsiveness, gait flabby, sternutation, and emiction incontinence were observed 10 min after dosing in 5/5 females, while one animal returned to normal 3 days after treatment.
464 mg/kg bw: Salivation, unresponsiveness, gait flabby, sternutation, and emiction incontinence were observed 10 min after dosing in 5/5 females.

Based on the mortalities, the LD50 value was calculated to be 110 (55 - 220) mg/kg bw for female rats.
Based in the results of this acute oral toxicity study, the three doses (0.88, 4.40 and 22.00 mg/kg bw/day) were selected for the teratogenicity study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: for general health condition

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded on gestation Day 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by CO2
- Organs examined: uterus
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 early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead/live fetuses: Yes
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all fetuses
- Soft tissue examinations: Yes, approximately 1/3 of the fetuses
- Skeletal examinations: Yes: approximately 2/3 of the fetuses
- Head examinations: No
- Anogenital distance of all live rodent pups: No
- Other: Sex, body weight, body length and tail length of live fetuses were recorded. 1/3 of the fetuses from each litter was taken and fixed in Bouin's solution for 2 weeks. Subsequently, the splanchnic malformations of fetus was examined. For the other 2/3 fetuses from each litter, the skin, organs and fat tissue were removed from the body. The skeleton was fixed in 90% ethanol solution at least 12 h and then put in 1% sodium hydroxide solution 3 - 5 days until the muscle became transparent and skeleton muscle could be clearly observed. The skeleton was stained with alizarin red solution until skeleton became red and then changed to transparent solution I for 1 - 2 days, followed by transparent solution II for 1 - 2 days. In the end skeleton was changed to transparent solution III for 1 - 2 days and after the skeleton had been stained red and the colour on the soft tissue faded, the sample was put into glycerol and skeletal malformations was examined.
Statistics:
All ratios were analyzed by chi-square test. Study measurements were analyzed by ANOVA or nonparametric statistics. Fetus body length, tail length and body weight were compared by t test. The data of fetuses was analyzed in the unit of litter．
Indices:
No indices were calculated.
Historical control data:
Historical control data were not provided.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities were detected in the female rats during the study.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in any group during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
0.88 mg/kg bw/day: No body weight changes were observed when compared to the control group.
4.40 mg/kg bw/day: No body weight changes were observed when compared to the control group.
22.00 mg/kg bw/day: The body weights of the pregnant rats were reduced during Day 8 until 20 compared to the control animals. The reduction in body weight reached statistical significance on day 20 compared to the control group (approximately -4%).

For detail, please refer to the attached background material 1.
Food consumption and compound intake (if feeding study):
not examined
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
Endocrine 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):
There were no statistically significant differences in uterus weight in any of the treatment group when compared to the control group. For detail, please refer to the attached background material 2.
Gross pathological findings:
not examined
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:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistically significant differences in the number of blastocyst implantation was observed in any of the treatment groups when compared to the control group.
For detail, please refer to the attached background material 2.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the number of absorbed of fetuses in any treatment group when compared to the control group.
For detail, please refer to the attached background material 2.
Dead fetuses:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the number of dead fetuses in any treatment group when compared to the control group.
For detail, please refer to the attached background material 2.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The study was conducted with pregnant rats (23 females in the treatment groups and 20 females in the control group). Therefore, no differences in the number of pregnant rats was observed.
For detail, please refer to the attached background material 2.
Other effects:
no effects observed
Description (incidence and severity):
Corpus lutea:
There was no statistically significant difference in the number of corpus lutea in females of any treatment group when compared to the control group.
For detail, please refer to the attached background material 2.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
4.4 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at this dose level.
Dose descriptor:
LOAEL
Remarks:
systemic toxicity
Effect level:
22 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
22 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No adverse effects in regard to maternal developmental toxicity observed up to the highest dose tested.

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the fetal body weights of the treatment groups when compared to the control group. For detail, please refer to the attached background material 2.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There was no statistically significant difference in the number of live fetuses of any treatment group when compared to the control group.
For detail, please refer to the attached background material 2.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex ration male:female was 100:80, 100:91, 100:96 and 100:105 in the control, low-, mid- and high dose group, respectively. The difference is not considered to be treatment-related as the largest difference was observed in the control group.
For detail, please refer to the attached background material 2.
Changes in litter size and weights:
not examined
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were observed in any of the treatment group. For detail, please refer to the attached background material 3.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Observed skeletal malformations were as follows:
Control: 5/169 (2.96%) rib malformations, 84/169 (49.70%) sternum ossification, rudimentary or not ossified and 4/169 (2.37%) thoracic vertebra malformation
0.88 mg/kg bw/day: 10/179 (5.59%) rib malformations, 91/179 (50.84%) sternum ossification, rudimentary or not ossified and 1/179 (0.56%) thoracic vertebra malformation
4.40 mg/kg bw/day: 10/186 (5.37%) rib malformations, 99/186 (53.23%) sternum ossification, rudimentary or not ossified and 7/186 (3.76%) thoracic vertebra malformation
22.00 mg/kg bw/day: 17/179 (9.50%) rib malformations, 125/179 (69.83%) sternum ossification, rudimentary or not ossified and 17/179 (9.50%) thoracic vertebra malformation
For detail, please refer to the attached background material 3.

No statistically significant differences in skeletal malformations were observed in fetuses treated with 0.88 or 4.40 mg/kg bw/day compared to the control group. In the high dose group, the fetus ratio of rib malformations and sternum ossification rudimentary or not ossified and thoracic vertebra malformation and total skeletal malformations were higher than in the control group. The type of rib and thoracic vertebra malformations were not specified, and no individual data was given. Furthermore, variations and malformations were not listed separately and therefore it is possible that variations (structural change considered to have little or no detrimental effect; maybe transient and may occur relatively frequently in the control population) were presented as malformations (structural change considered detrimental to the animal and is usually rare). The control group prevalence was high and no historical control data range was included. There was no clear dose-response for either the rib malformation or thoracic vertebra malformations and the incidence of sternum ossification rudimentary or not ossified was very high in the control, which would lead towards a conclusion that this effect was a variation rather than a malformation. Furthermore, the acute oral toxicity study performed as a dose-range finder showed the LD50 value was 55 – 220 mg/kg bw, with severe adverse effects and mortality observed from 46.6 mg/kg bw. The highest dose group in the developmental toxicity study was approximately half of the dose that caused mortality in the range-finding study. Therefore, it is likely that the dams were systemically affected by the test substance and the observed effects on the offspring are considered to be secondary to the maternal systemic effects.
Visceral malformations:
no effects observed
Description (incidence and severity):
No splanchnic malformations were observed in the treatment groups. For detail, please refer to the attached background material 3.
Other effects:
no effects observed
Description (incidence and severity):
Fetus body length:
There was no statistically significant difference in the body length of fetuses of any treatment group when compared to the control group.

Fetus tail length:
There was no statistically significant difference in the tail length of fetuses of any treatment group when compared to the control group.

For detail, please refer to the attached background material 2.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
4.4 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Effect level:
22 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Remarks on result:
other: adverse effects observed were secondary to maternal systemic toxicity.

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: rib
skeletal: vertebra
Description (incidence and severity):
Increased incidences of rib malformations, sternum ossification rudimentary or not ossified and thoracic vertebra malformations.

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
22 mg/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:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The study was conducted according to the Guidelines for the testing of chemicals section 4: Health Effects (Ministry of Environmental Protection of People's Republic of China) and comparable to OECD guideline 414 under non-GLP conditions. There were major deviations from the current OECD guideline: dams were exposed on gestation Day 6-15, the thyroid gland was not weighed or histopathologically assessed, no thyroid hormone levels were measured, 1/3 fetuses from each litter were examined for soft tissue alterations and all fetuses per litter were examined for skeletal alterations, anogenital distance was not measured, no individual data or raw data was included in the study report, and the developmental effects were not specified as variations and malformations. Under the conditions of this study, the maternal NOAEL for general toxicity was 4.40 mg/kg bw/day based on a statistically significant reduction in body weight. The fetal developmental NOAEL was 4.40 mg/kg bw/day. At 22.00 mg/kg bw/day, statistically significantly increased incidences of rib malformations, sternum ossification rudimentary or not ossified and thoracic vertebra malformations were observed compared to the control animals. However, variations and malformations were not listed separately and therefore it is possible that variations were presented as malformations. The type of variations/malformation was not specified. The control group prevalence was high and no historical control data range was included. There was no clear dose-response for either the rib malformation or thoracic vertebra malformations and the incidence of sternum ossification rudimentary or not ossified was very high in the control, which would lead towards a conclusion that this effect was a variation rather than a malformation. Furthermore, the acute oral toxicity study performed as a dose-range finder showed the LD50 value was 55 – 220 mg/kg bw, with severe adverse effects and mortality observed from 46.6 mg/kg bw. The highest dose group in the developmental toxicity study was approximately half of the dose that caused mortality in the range-finding study. Therefore, it is likely that the dams were systemically affected by the test substance and the observed effects on the offspring are considered to be secondary to the maternal systemic effects.