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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:
May 24 – Jun. 10, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 25, 2018
Deviations:
yes
Remarks:
The purpose of this study was to determine the dose range of the test substance for prenatal developmental toxicity study. Less parameters compared to a full study were examined.
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Allyl (cyclohexyloxy)acetate
EC Number:
272-657-3
EC Name:
Allyl (cyclohexyloxy)acetate
Cas Number:
68901-15-5
Molecular formula:
C11H18O3
IUPAC Name:
allyl (cyclohexyloxy)acetate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: ORIENTBIO INC., Republic of Korea
- Age at study initiation: at start of mating: males: 11 weeks, females: 10 weeks; at start of administration: 11 weeks (females only)
- Weight at start of mating: males 385.3 – 415.0 g, females 212.4 – 248.1 g; at start of administration: 245.8 – 290.3 g (females only)
- Housing: Stainless wire mesh cages, 260W×350D×210H (mm), (excluding use of polycarbonate cages), Polycarbonate cages, 260W×420D×180H (mm), (from late stage of pregnancy to necropsy)
-Number of animals per cage: One–two animals (during the quarantine-acclimation
period), One male and one female (during the mating period), One animal (during the gestation period)
- Diet: ad libitum, Powder feed rodent chow, (LabDiet® CERTIFIED RODENT DIET 5002, powder type)
- Water: Public tap water in Cheongju-si was filtered and irradiated by ultraviolet light and
provided ad libitum.
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Analysis of feed
The certificate of feed analysis was provided by the manufacturer, LabDiet® . The results of feed analysis met the allowable standard of this facility.
Analysis of drinking water
Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year by the Research Institute of Health & Environment, ChungBuk (184, Osong saengmyeong 1 (il)-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea) according to the Regulation of Quality Criteria for Potable Water and Test (Ministry of Environment Ordinance No. 792, Revision Dec. 26, 2018). The results of water analysis met allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 22.5
- Humidity (%): 44.9 - 65.2
- Air changes (per hr): 10 - 15
- Photoperiod: 12 hour light/dark cycle

IN-LIFE DATES: From: May 12, 2020 To: Jun. 8 – 11, 2020

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION
The required amount of the test substance was weighed and placed in a bottle. The required amount of vehicle (required amount: 10 mL of vehicle for 1 kg of powder feed), corn oil, was added and mixed using a vortex mixer until dissolved.
The required amount of powder feed except for the amount of the test substance was weighed. The required amount of the test substance formulation and a small amount of powder feed were mixed in a bottle. And then, the mixture was placed in a ball mill (Universal Ball Mill, UBM-200L, Intec System, Republic of Korea) and residual powder feed was added and mixed using the ball mill for approximately 5 – 10 minutes to yield the desired concentration. The required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5 – 10 minutes for the control group. The dosing feed was stored in a refrigerator within 14 days and used at room temperature within 7 days.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
from Gestation day (GD) 5 to GD 19
Frequency of treatment:
daily
Duration of test:
Mating period: May 18 – 22, 2020
Administration period: May 24 – Jun. 10, 2020
Necropsy and Caesarean section: Jun. 8 – 11, 2020 (GD 20)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
27.2 mg/kg bw/day
Remarks:
mean total intake
equivalent to 0.03 % in diet
Dose / conc.:
84.5 mg/kg bw/day
Remarks:
mean total intake
equivalent to 0.1 % in diet
Dose / conc.:
235.8 mg/kg bw/day
Remarks:
mean total intake
equivalent to 0.3 % in diet
No. of animals per sex per dose:
6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Mated females were weighed and equentially assigned to the control and dosing groups (6 animals per group, total 4 groups) based on stratified body weights in an attempt to equalize body weights between the groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were observed for mortality, general condition, clinical signs, excretion, abortion and premature birth at least once daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes, see above

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured on Days 0, 3, 5 – 19 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

GROSS PATHOLOGY: Yes
All animals were sacrificed on Gestation Day 20 by exsanguination from the abdominal aorta under isoflurane anesthesia. Complete gross postmortem examinations were conducted on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.
The following organs of all pregnant animals were harvested and wet weight was measured, and the relative ratio of organ weight to terminal body weight of dams (excluding gravid uterus weight) was calculated:
Gravid uterus, Thyroid gland, Liver

HISTOPATHOLOGY: Yes
At necropsy, the following organs and tissues from all pregnant animals were
harvested and preserved in 10% neutral buffered formalin.
· Brain
· Pituitary gland
· Ovary
· Uterus
· Thyroid gland
· Liver
· Organs/tissues with gross lesions
Histopathological evaluation on the samples of the following organs and tissues was performed:
– Liver and thyroid from all pregnant animals
– Organs with gross lesions

Caesarean section
All animals were subjected to a full external examination on gestation day 20. The ovaries and uteri of pregnant females were removed, and implantation, early and late resorptions and dead/live fetuses were examined and the number of pregnancy corpora lutea was counted. The pre- & post-implantation rates were calculated.
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
Blood sampling:
- Plasma: No
- Serum: No
Fetal examinations:
- External examinations: Yes: all per litter

Statistics:
The statistical analysis of this study was conducted using the SAS program Version 9.3, SAS Institute Inc., U.S.A.).
Indices:
· Pre-implantation loss rate (%) = ((Number of corpora lutea – Number of implantations) / Number of corpora lutea) ×100

· Post-implantation loss rate (%) = ((Number of implantations – Number of live fetuses) / Number of implantations) × 100

· Sex ratio = Number of live male fetus / Number of live female fetus

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 0.1% group, statistically significant decreased mean body weights on GD 14 and GD 20 (93% and 92% of the control, respectively) and decreased body weight gain during the treatment period (from GD 5 to GD19, 86% of the control) were observed. In the 0.3% group, statistically significant decreased mean body weights over the period from GD 8 to GD 20 (ranging from 84% to 87% of the control) and decreased body weight gain during the treatment period (from GD 5 to GD 19, 53% of the control) were observed. In addition, a body weight loss was observed in the early stage of pregnancy, especially at GD 8. These changes were observed along with decreased food consumption in the 0.1 and 0.3% group beginning from the GD 6 to GD 15, and it was considered to be treatment-related as they were observed in a dose – dependent manner.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 0.3% group, a statistically significant decrease in food consumption was observed on GD 6 and GD 9 (17% and 71% of the control, respectively).
A decrease in food consumption was observed in the 0.1% and 0.3% groups when compared to the control group from GD 6 to GD 15 (74% – 90%, 17% – 91% of the control, respectively).
A decrease in the relative food consumption was observed in the 0.1% and 0.3% groups when compared to the control group on GD 6 and GD 9 (79% and 87%, 20% and 82% of control, respectively).
These changes in food consumption were considered treatment-related and body weight and body weight gain were also decreased during the same period.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All microscopic findings (e.g. inflammatory cells in liver) seen in the liver and thyroid gland did not show significant difference in the incidence compared to controls or were normal background lesions observed in this facility or were found with low/ isolated frequency.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All microscopic findings (e.g. cysts in thyroid gland) seen in the liver and thyroid gland did not show significant difference in the incidence compared to controls or were normal background lesions observed in this facility or were found with low/ isolated frequency.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
84.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: equivalent to 0.1 % in diet
Key result
Dose descriptor:
NOAEL
Effect level:
27.2 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects
Remarks on result:
other: eqivalent to 0.03 % in diet

Maternal abnormalities

Key result
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
External malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
235.8 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
other: equialent to 0.3 % in diet

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Applicant's summary and conclusion

Conclusions:
Based on the dose range finding study results 0.2% was selected as the high dose of the main study. The mid and low doses were selected to be 0.07 and 0.02 %, respectively.
Executive summary:

The purpose of this study was to determine the dose range of the test substance for prenatal developmental toxicity study by evaluating the effects on dams and embryo-fetal development when administered orally (dietary) to pregnant Sprague-Dawley (SD) rats from implantation to closure of the hard palate (from Day 5 to Day 19 of Gestation). The test doses were 0, 0.03, 0.1 and 0.3% (0, 27.2, 84.5 and 235.8 mg/kg bw/d).


A decrease in body weight, body weight gain and food consumption was observed at 0.1% and 0.3% during the treatment period. Based on the dose range finding study results 0.2% was selected as the high dose of main study. The mid and low doses were selected to be 0.07 and 0.02%, respectively.