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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
Study period:
2020-09-16 to 2022-01-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018-06-25
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Regulation on Test Methods for Chemical Substances” Notification No. 2020-28 National Institute of Environmental Research, Republic of Korea
Version / remarks:
2020-08-19
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Remarks:
(Crl:CD(SD)), SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: at the start of mating: 11 weeks (males), 10 weeks (females); at the start of administration: 11 weeks (females)
- Weight at study initiation: at the start of mating: 336.3 – 420.1 g (males), 213.7 – 254.7 g (females); at the start of administration: 231.3 – 309.1 g (females)
- Housing: 1-2 animals were housed together during acclimatation period, 1 male and 1 females together during mating period and females were housed singly during gestation period. Animals were housed in stainless wire mesh cages, 260W×350D×210H (mm) and in polycarbonate cages, 260W×420D×180H (mm) from late stage of pregnancy (GD 16) to necropsy
- Diet: Ad libitum (LabDiet® CERTIFIED RODENT DIET 5002, powder type)
- Water: Ad libitum (tap water, filtered and irradiated by ultraviolet light)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 23.4
- Humidity (%): 52.4 – 61.7
- Air changes (per hr): 10 – 15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (150 – 300 Lux)

IN-LIFE DATES: From day 5 to day 19 of gestation (15 days)

Administration / exposure

Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was mixed with the required amount of vehicle (required amount: 10 mL of corn oil for 1 kg of powder feed) 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 and then, the mixture was placed in a ball mill and residual powder feed was added and mixed for approximately 5 – 10 minutes. 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.


DIET PREPARATION
- Mixing appropriate amounts with: Powder feed rodent chow (LabDiet® CERTIFIED RODENT DIET 5002, powder type, LabDiet® , U.S.A., Lot No: 5002, 25LB SEP04193A)
- Storage temperature of food: The dosing feed was stored in a refrigerator within 14 days and used at room temperature within 7 days.


VEHICLE
- Justification for use and choice of vehicle: Solubility properties
- Concentration in vehicle: 0.2%, 0.02% and 0.07%
- Batch No: MKCK6411
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis for homogeneity and stability: Homogeneity and stability analyses were conducted in a separate study by GC (Study No.: B16569). The 0.01% and 0.3% dosing formulations were confirmed to be homogenous and stable for 7 days at room temperature and for 14 days under refrigeration.

Verification of dose level concentrations: Analyses of the dosing formulations were conducted using GC using the method described in Study No.: B16569. Samples were taken three times from the middle of each dosing formulation and analyzed for verification of dose level concentration prior to the first dosing. As a result, the accuracies at 0.02, 0.07 and 0.2% were 97.15, 92.21 and 94.75% prior to dosing, respectively. These results were within the acceptable range (range: ±15% of nominal values).
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: 4 days
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: Vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From gestation day 5 to gestation day 19 (15 days)
Frequency of treatment:
Continuously via feed (ad libitum)
Duration of test:
From gestation day 5 to gestation day 19 (15 days)
Doses / concentrationsopen allclose all
Dose / conc.:
175.9 mg/kg bw/day (actual dose received)
Remarks:
Mean value, corresponds to 0.2% substance concentration in feed
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
Mean value, corresponds to 0.07% substance concentration in feed
Dose / conc.:
16.1 mg/kg bw/day (actual dose received)
Remarks:
Mean value, corresponds to 0.02% substance concentration in feed
No. of animals per sex per dose:
20 animals per sex per dose. 2 animals of the high dose group were not pregnant and excluded from the evaluation
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a result of the dose range finding study (Study No.: B20070, see supporting study), no deaths were observed at doses of 0.1% and 0.3%, but decreased body weight and food consumption. Therefore, based on the dose range finding study, 0.2% was selected as the high dose of this study. The mid and low doses were selected at 0.07% and 0.02%, respectively.
- Time of day for dam blood sampling: Blood samples from the abdominal aorta of animals were taken within a short time (two hours) on the morning of the day of necropsy

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations included: Mortality, moribundity, general appearance, abortion and premature birth

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: On days 0, 3, 5, 8, 11, 14, 17, 19 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Gross post mortem examinations, including the external surface and internal organs. The wet weight of gravid uterus and liver was taken and the relative ratio of organ weight to terminal body weight of dams (excluding gravid uterus weight) was calculated. Thyroid gland with parathyroid gland (paired) was weighed after the fixation.

OTHER: THYROID HORMONE ANALYSIS
- The following parameters from separated serum were analyzed using an immunoassay analyzer: Triiodothyronine (T3), Total thyroxine (T4) and Thyroid stimulating hormone (TSH) using a chemical luminescence immunoassay
- Time schedule: Blood samples from the abdominal aorta of animals were taken on the morning of the day of necropsy

HISTOPATHOLOGY
At necropsy, the following organs and tissues from all pregnant animals were harvested and preserved in 10% neutral buffered formalin: Brain, Ovary, Thyroid gland, organs/tissues with gross lesions, pituitary gland, uterus, liver, parathyroid gland. Histopathological evaluation was performed on the following organs: Liver, thyroid gland, organs/tissues with gross lesions
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
- Other: Number of dead/live fetuses
Blood sampling:
- Plasma: No
- Serum: Yes
Fetal examinations:
- External examinations: Yes (all per litter): Eyes, ears, mouth, palate, absent of limbs and tail, position, size and shape were examined.
- Soft tissue examinations: Yes (half per litter)
- Skeletal examinations: Yes (half per litter)
- Head examinations: No data
- Anogenital distance of all live rodent pups: Yes
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 fetuses / Number of total live fetuses× 100
- Anogenital index = Anogenital distance (AGD) / ∛body weight
Historical control data:
Historical control data were provided to allow comparison with concurrent controls (see "Attached background material")

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no deaths or abnormalities of clinical signs in animals in the control, 0.02, 0.07 and 0.2% groups during the observation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
0.2% group: Statistically significant decrease in body weight from GD 8 to GD 20 (88.3% – 90.9% of control). A statistically significant decrease in body weight gain was observed between GD 8 and GD 20 (except on GD 11 and 17, 36.6% – 81.4% of control).

0.07% group: Statistically significant decrease in body weight from GD 8 to GD 20 (94.1% – 95.6% of control). A statistically significant decrease in body weight gain was observed between GD 8 and GD 14 (46.9% and 74.5% of control, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
0.2% group: Statistically significant decrease in food consumption on GD 19 (81.8% of control).

Other statistical significances in food consumption and relative food consumption were considered not to be test substance-related changes since there was no correlation to the body weight changes.
Food efficiency:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related change in thyroid hormone was observed in the study.

For T3 and T4, a statistically significant increase observed in the 0.07 and 0.2% groups was considered not to be test substance-related because it did not show a dose-response relationship and was not accompanied by any correlated changes in organ weight or histopathology.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
0.2% group: Statistically significant decrease in the absolute but not relative organ weight of the liver. A statistically significant increase in the relative organ weight of the gravid uterus was observed. As changes were small and the absolute weight was a little lower than that of the control and no clear dose-dependency was noted for absolute and relative weights, this variation was considered not being indicative of a test-related effect.

0.07% group: Statistically significant decrease in the absolute but not relative organ weight of the liver
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination did not reveal test substance-related changes in all pregnant or non-pregnant females in any test substance-treated groups.

All microscopic findings seen in the liver and thyroid gland did not show significant difference in the incidence compared to controls or were normal background lesions observed at this facility or were found with low/isolated frequency.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test substance-related effects were observed in pre-/post-implantation loss rates in the study.
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
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
0.2% group: 2 females were found non-pregnant at caesarean section. This finding was not chosen test item-related.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
16.1 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
0.2% group: Statistically significant decrease in body weight of male and female fetuses (95.0% and 94.2% of control, respectively)
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
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No test substance-related effect was observed in the AGD index of male and female fetuses in the study.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test substance-related effects in external findings of fetuses and placentas were observed in the study.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related effects were observed with regard to skeletal malformations.

Skeletal malformations observed were considered not to be test substance-related effects because these were not statistically significant and occurred only in the low dose group.

No test substance-related effects were observed with regard to skeletal variations. The skeletal variations observed in the control, 0.02, 0.07 and 0.2% groups were considered not to be test substance-related effects because these were not statistically significant and/or were not dose-dependent and/or inside the historical control range.

The number of ossified proximal phalanges in the forelimb were observed with a statistically significant decrease in the 0.02. 0.07 and 0.2% groups. This finding was considered not to be test substance-related because it did not show a dose-response relationship and was within the historical control range. Furthermore, it might be considered as secondary effect to the decreased fetal and maternal body weight at 0.2%.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects were observed in the visceral examination of fetuses in the study.

Malformations observed consisted of one case each of misshapen brain and cleft palate and were not statistically significant and occurred in the control and low dose groups, respectively, and, therefore, were not considered as test substance-related effects.

Variations observed, including dilated ureter, convoluted ureter, dilated renal pelvis in kidneys, and malposition in umbilical artery, were not considered as test substance-related effects.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
This Prenatal Development Toxicity Study according to OECD guideline 414 study was carried out to investigate the potential adverse effects of test substance on pregnant females and embryo-fetal development in rats. The test substance was orally (dietary) administered to pregnant females (20 animals per group) from implantation to closure of the hard palate (from day 5 to day 19 gestation, total 15 days) at doses of 0 (vehicle control), 0.02 (corresponding to 16.1 mg/kg bw/day), 0.07 (corresponding to 60 mg/kg bw/day) and 0.2% (corresponding to 175.9 mg/kg bw/day). In pregnant females, suppressed body weight and decreased body weight gain were observed in the 0.07% and 0.2% groups and decreased food consumption was observed in the 0.2% group. In the fetuses, decreased body weight was observed in the 0.2% group. Therefore, the no-observed-adverse-effect level (NOAEL) for pregnant rats is considered to be 0.02% (16.1 mg/kg bw/day). The NOAEL for embryo-fetal development is considered to be 0.07% (60 mg/kg bw/day).
Executive summary:

The purpose of this Prenatal Development Toxicity Study according to OECD guideline 414 and GLP was to investigate the potential adverse effects of test substance on pregnant females and prenatal development when administered orally (dietary) to the pregnant Sprague-Dawley rats (20 animals per group) from implantation to the day before necropsy (from day 5 to day 19 of gestation, total 15 days). The administration was conducted at doses of 0 (vehicle control), 0.02, 0.07 and 0.2% (0, 16.1, 60.0, and 175.9 mg/kg/day, respectively). Evaluated parameters included clinical signs, body weight, food consumption, thyroid hormone analysis, organ weights, gross pathology, histopathology, caesarean section, measurement of body weights of live fetuses and placenta weights, anogenital distance (AGD) of fetuses, and external, visceral and skeletal examinations of fetuses.


 


No clinical signs or deaths related to the test substance occurred in the treatment groups during the study period. In the 0.07% group, a statistically significant decrease in body weight (94.1-95.6% of control) was observed over the period of gestation day (GD) 8 to 20, and a statistically significant decrease in body weight gain (46.9 and 74.5% of control, respectively) was observed on GD 8 and GD 14. In the 0.2% group, a statistically significant decrease in body weight (88.3-90.9% of control) was observed over the period of GD 8 to GD 20, and a statistically significant decrease in body weight gain was observed over the period of GD 8 to GD 20 (except on GD 11 and 17, -36.6%-81.4% of control). A statistically significant decrease in food consumption (81.8% of control) was observed on GD 19.


No test substance-related changes were observed in thyroid hormone (T3, T4 and TSH) levels, caesarean section results (pre-/post-implantation loss rates, sex ratio), absolute and relative organ weights, gross pathology, and histopathology during the study. In the fetuses, statistically significant decreased body weight in males and females (95.0% and 94.2% of control, respectively) was observed in the 0.2% group. No test substance-related changes were observed in the placenta weight, external examination of fetuses and placentas, AGD, skeletal and visceral examination at any dose tested.


 


In conclusion, oral (dietary) administration of the test substance to pregnant rats resulted in a decrease of the maternal weight at 0.07% and a decrease of maternal weight and food consumption at 0.2%. The decrease of fetal weight at 0.2% was considered a secondary effect of decreased maternal weight at this dose. In the present experimental conditions, the no-observed-adverse-effect level (NOAEL) of the test substance is considered to be 0.02% for pregnant females and 0.07% for embryo-fetal development in rats.