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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-04-29 to 2021-XX-XX. The final study period will be added as soon as the final report is available.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2020

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
1998-09-21, adopted 2018-06-25
Deviations:
no
GLP compliance:
yes
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 species / strain selection:
Sprague-Dawley rats are commonly used in toxicity studies, having a large historical control data base.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 186.5 to 214.2 g (males) and 147.8 to 180.1 g (females)
- Fasting period before study: No
- Housing: Singly in stainless wire mesh cages, 260Wx350Dx210H (mm)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 7 days

DETAILS OF FOOD AND WATER QUALITY: The certificate of feed analysis was provided by the manufacturer, LabDiet®. The results of feed analysis met the allowable standard of this facility. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year 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 the allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5–25.7
- Humidity (%): 42.8–83.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 91 or from day 1 to day 119 (recovery groups)

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral (dietary) route was selected to assess the toxicity by oral (dietary) exposure of the test substance.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
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. Then, the mixture was placed in a ball mill and residual powder feed was added and mixed for approximately 5 – 10 minutes to yield the desired concentration. For the control group, 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.

DIET PREPARATION
- Rate of preparation of diet: Every 7-14 days
- Mixing appropriate amounts with: Powder feed rodent chow (LabDiet® CERTIFIED RODENT DIET 5002, powder type)
- Storage temperature of food: At approx. 4°C for 14 days or at room temperature for 7 days

VEHICLE
- Justification for use and choice of vehicle: Solubility properties
- Concentration in vehicle: 10% (for a final concentration of 0.1%), 3% (for a final concentration of 0.03%) and 1% (for a final concentration of 0.01%)
- Batch No: MKCH1635, MKCK6411
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses for homogeneity and stability: Homogeneity and stability were analyzed in a separate study (Study No: B16569).Tthe 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: Dose level concentration were analyzed in a separate study (Study No: B16569) using Gas Chromatography. Samples were taken three times from the middle of each dosing formulation and analyzed for verification of dose level concentration prior to dosing and at Week 13 . As a result, the accuracies at 0.01, 0.03 and 0.1% were 106.10, 102.13 and 97.72% prior to dosing and 109.20, 103.67 and 100.80% at week 13, respectively. These results were within the acceptable range (range: +/-15% of nominal values).
Duration of treatment / exposure:
90 days (all dose and control groups) or 118 days (recovery groups)
Frequency of treatment:
Continously
Doses / concentrationsopen allclose all
Dose / conc.:
75.7 mg/kg bw/day (actual dose received)
Remarks:
Determined for females, corresponding to 0.1% of the test substance in feed
Dose / conc.:
67.6 mg/kg bw/day (actual dose received)
Remarks:
Determined for males, corresponding to 0.1% of the test substance in feed
Dose / conc.:
23.5 mg/kg bw/day (actual dose received)
Remarks:
Determined for females, corresponding to 0.03 % of the test substance in feed
Dose / conc.:
20.1 mg/kg bw/day (actual dose received)
Remarks:
Determined for males, corresponding to 0.03 % of the test substance in feed
Dose / conc.:
7.9 mg/kg bw/day (actual dose received)
Remarks:
Determined for females, corresponding to 0.01% of the test substance in feed
Dose / conc.:
7 mg/kg bw/day (actual dose received)
Remarks:
Determined for males, corresponding to 0.01% of the test substance in feed
No. of animals per sex per dose:
10 animals + 5 recovery animals/sex/group in the control and high dose groups and 10 animals/sex/group in the low and mid dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In dose range finding study of the OECD 407 (Study No.: B16567), the body weight was significantly reduced at 0.3 % (21 % in males, 14 % in females). Also the body weight gain showed a significant decrease of more than 40 %. Therefore, 0.1 % was selected as the high dose level for the main study of the OECD 407 (Study No.: B16568). This 4-week repeated dose toxicity study showed a significant decrease of body weight (15%) along with a significant decrease of the body weight gain of more than 30 %. This was regarded as an early indication of toxicity. Taken together the DRF and the main study, higher doses than 0.1 % were not considered suitable for the longer exposure time of the OECD 408 and the high dose level for the 90-Day repeated dose toxicity study was therefore set to 0.1 %. Mid and low dose levels were selected at 0.03 and 0.01%, respectively.
- Fasting period before blood sampling for clinical biochemistry: Approximately 18 hours
- Rationale for selecting satellite groups: Recovery groups (control group and high dose group) were were kept without treatment for 28 days after the last dosing day to examine the reversibility of potential effects.
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
Not included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily (for clinical signs) and twice daily (for mortality and moribundity)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to dosing and once weekly
- Detailed clinical observations checked in table 1 were included.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing on Day 1, once a week during the dosing and recovery periods and on the day of necropsy. Body weight data recorded on the day of necropsy was not included in the evaluation of body weights since these data are body weights of fasting animals.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time x 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to dosing (all animals) and at Week 13 (all animals in the control and high dose groups of the main group)
- Dose groups that were examined: Control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 2+3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 4 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: On the day of necropsy
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: At week 13 (all animals of the main group) and at recovery week 4 (recovery groups)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: While fresh urine (3-hour) was collected, feeding and dosing were not performed.
- Parameters checked in table 5 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: On the main group animals at Weeks 12–13 and on the recovery group animals at Recovery Weeks 3–4.
- Dose groups that were examined: All dose groups and the control group were examined.
- Battery of functions tested: Sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 6)

HISTOPATHOLOGY: Yes (see table 7)
Optional endpoint(s):
Optional endpoints: Yes

OBSERVATION OF ESTRUS CYCLE
Observation of the estrus cycle stage for all females in the main and recovery group were conducted in the morning on the day of necropsy. Prepared smears of the vaginal mucosa were stained with Diff Quick stain. Stained vaginal mucosa smears were examined using light microscopy.

EXAMINATION OF SPERM
Examination of sperm was evaluated for all surviving males in the main group and all males in the recovery group. The left epididymis was weighed.

EXAMINATION OF SPERM CAUDAL EPIDIDYMAL SPERM
The epididymides were weighed. At necropsy, the left caudal epididymis was quickly incised, weighed and placed in DPBS (Dulbecco’s phosphate buffered saline) containing 1% BSA (Bovine Serum Albumin). Sperm was incubated for approximately 3 to 10 minutes in 1% BSA-DPBS culture medium (at approximately 37°C). Samples were diluted to the appropriate concentrations, placed on glass slides and evaluated for sperm motility using a sperm analyzer. Parameters for sperm motility are shown in table 8. Sperm samples were smeared on the glass slides, stained with Diff-Quick and examined for morphology using a light microscope. The number of abnormal sperm was counted from approximately 200 sperms per smear.

EXAMINATION OF CAUDAL EPIDIDYMAL SPERMATID HEAD COUNTS
The left epididymis used for sperm motility test was wiped off all external particulate matter and refrigerated for each animal. The tail of the epididymis was incised, weighed, and the membrane was removed. The epididymis were placed in 8 mL of distilled water and homogenized. The total sperm counts for each animal was determined by counting homogenization-resistant sperm heads using a microscope. The number of sperm per 1 g of the left epididymis was calculated.
Statistics:
Main groups: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, examination of sperm (except for sperm malformation) and organ weights were analyzed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data; then, if significant, Dunnett’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
Main group of sperm malformation: Kruskal-Wallis test was employed on heterogeneous data; then, if significant, Steel’s test was employed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).

Recovery group: the data of body weight, food consumption, functional observations (hindlimb landing foot splay, grip strength and motor activity), urine volume, hematology, clinical chemistry, examination of sperm and organ weights were analyzed utilizing Folded-F test for homogeneity of variance (significance level: 0.05). Student’s t-test was employed on homogeneous data (significance level: 0.05) or Aspin-Welch t-test was employed for heterogeneous data (significance level: 0.05) for verifying significance (significance levels: 0.05 and 0.01, two-tailed).

The analyses are considered to be appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
0.1% group: One male animal with overgrown teeth (day 50), and one male with loss of teeth on day 116

0.03% group: One female animal showing loss of teeth (day 26).

Loss of teeth was not considered to be a test substance-related effect since it was observed in only one animal of each sex and thus judged to be incidentally.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
0.1% group: Statistically significant decreases in body weights of males from Week 1 to Week 13 and of females from Week 5 to Week 13 (except for Week 7) when compared to the control group. Neither during nor at the end of the recovery period, there were statistically significant differences in body weights in males and females when compared to the control groups.

Statistically significant decreases in body weight gain in males from Week 1 to Week 4 (except for Week 3) and Week 7.

The mean body weights at the end of the dosing period (Week 13) and after the recovery period (Week 17) were 14.2% and 7.6% (males) or 7.9% and 1.1% (females) lower than those of the control groups, respectively.

0.03% group: Statistically significant decreases in body weight gain in females at Week 11 when compared to the control group.

The decrease in body weights was correlated with the reduced food consumption in males and females in the 0.1% group. At the end of the recovery period, body weight and food consumption changes were observed to have the tendency of recovering.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
0.1% group: Statistically significant decreases in the relative food consumption in males at Week 1, 2, 4, 5 and 7 and in females at Weeks 5 and 9.

0.03% group: Statistically significant decreases in the relative food consumption in males at Weeks 1, 2, 4 and 7 and in females at Week 5.

0.01% group: . Statistically significant decreases in the relative food consumption in females at Weeks 9.

During the recovery period, there were statistically significant increases in the relative food consumption in males in the 0.1% group at Week 15 (recovery Week 2) when compared to the control group. At the end of the recovery period, food consumption changes had a tendency of recovering.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
All differences in hematology parameters were of small magnitude and even if statistically significant they were judged to be produced due to biological variation and are within the historical control range. In addition, the variations were not dose-depended or only observed in the recovery group.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
All differences in clinical chemistry parameters were of small magnitude and even if statistically significant they were judged to represent normal biological variations and were within the historical control range and/or without any histopathological correlate.). In addition, the variations were neither observed in both sexes nor confirmed in related parameters. Furthermore, some changes were observed in the recovery group only.
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
differences in urinalysis were of small magnitude thus judged to be of biological variation, considered to be unrelated to dosing of the test substance or because the individual data showed minor variations being within the historical reference range.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
If organ weight changes were noted, there were of small magnitude or within the historical control range. In addition, the variations were neither accompanied by morphological alteration (no histopathological changes) nor confirmed by related parameters. Furthermore, some changes were considered secondary effects caused by decreased body weights only.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All other microscopic findings in various organs and tissues were considered to be incidental, spontaneous, and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No effects on estrus cyclicity and sperm parameters were observed. All differences in sperm parameters were judged to be produced due to biological variation or were considered to be unrelated to dosing of the test substance based on small magnitude.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 67.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Preliminary NOAEL, as study not yet final
Key result
Dose descriptor:
NOAEL
Effect level:
>= 75.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Preliminary NOAEL, as study not yet final

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) of the test substance was 0.1%, corresponding to 67.6 mg/kg bw/day (males) and 75.7 mg/kg bw/day (females) under the conditions of this study.
Executive summary:

In a subchronic repeated dose toxicity study according to OECD guideline 408 and GLP, the cumulative toxicity of the test substance when administered orally (dietary) to 6-week-old Sprague-Dawley (Crl:CD(SD)) rats for 90 days was assessed.


 


A total of 4 groups were designated as follows:


Three groups receiving the test substance were designated as Groups 2, 3 and 4 at dose levels of 0.01, 0.03 and 0.1 % (7.0, 20.1 and 67.6 mg/kg/day for males, and 7.9, 23.5 and 75.7 mg/kg/day for females, respectively) along with a control group, Group 1 (the standard basal powder feed with corn oil), each consisting of 10 males and 10 females for main study. Five animals were added to each group for the recovery groups, control (G1) and high dose (G4) groups.


Evaluated parameters included clinical signs, detailed clinical sign observations, body weight, food consumption, functional observations, ophthalmological examination, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm, gross post mortem examination, organ weights and histopathological evaluations of selected tissues.


All animals survived the duration of the study. There were no effects on mortality.


No test substance-related toxic effects were noted in the results of body weight, clinical signs, detailed examinations of clinical signs, functional observations, urinalysis, hematology, clinical chemistry, observation of estrus cycle, examination of sperm, organ weights and necropsy.


Histopathologically, there were no test substance-related effects in males and females in the 0.1% groups.


 


Based on the results, the preliminary no observed adverse effect level (NOAEL) of the test substance is considered to be 0.1%, corresponding to 67.6 mg/kg bw/day (males) and 75.7 mg/kg bw/day (females) under the conditions of this study. The NOAEL might be updated after finalization of the study.

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