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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-10-27 to 2017-03-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016-07-29
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
not applicable

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxypropyl [(1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl] carbonate
EC Number:
607-858-0
Cas Number:
260781-16-6
Molecular formula:
C14H26O4
IUPAC Name:
2-hydroxypropyl [(1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl] carbonate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The current state of scientific knowledge does not provide acceptable alternatives, in vitro or otherwise, for the use of live animals to accomplish the purpose of this study. The rat is a universally used model for evaluating developmental and reproductive toxicity of various classes of chemicals, and for which there is a large historical database.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc. 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
- Age at receipt: males: approx. 7 weeks; females: approx. 9 weeks
- Age at study initiation: males: approx. 10 weeks; females: approx. 12 weeks
- Weight at study initiation: males: 319.8 - 428.3 g; females: 235.5-279.3 g
- Housing:
acclimatisation, pre-treatment, treatment, and post-mating: housed individually or in groups of 2 in stainless-steel cage (255 W x 465 L x 200 H mm)
mating: 1 male and 1 female/stainless-steel cage (255 W x 465 L x 200 H mm)
gestation: 1 mated female/poly sulfone cage (260 W x 420 L x 180 mm)
lactation: 1 dam with pups/poly sulfone cage (260 W x 420 L x 180 mm)
bedding material: sterilized aspen animal bedding material
- Diet (ad libitum): standard rat and mouse pellet diet (Lab Diet® #5053 PMI Nutrition International; irradiated by gamma-ray)
- Water (ad libitum): filtered, ultraviolet light-irradiated municipal tap water
- Acclimation period: 6 days
- Pre-treatment period before dosing started: 14 days (determination of oestrus cycle was conducted)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 - 20 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- test item was weighed and suspended in the vehicle and stirred with a stirrer in order to prepare the target concentration.
- formulation for high dose group was prepared first and then lower dose formulations were prepared by diluting the higher dose formulation with corn oil.
- dose formulations were prepared at least one time per week and stored at room temperature.

- dose was based on the most recently measured body weight (dose volume: 2 mL/kg).
- dose formulation was continuously stirred (before and throughout the dosing procedure).

VEHICLE
- Justification for use and choice of vehicle: corn oil was considered non-toxic at the applied volume of 2 mL/kg bw, and it has been used in previous studies since the test item can be suspended in this vehicle.
- Lot/batch no.: MKBW9504V
Details on mating procedure:
- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: 1 mated female/poly sulfone cage (260 W x 420 L x 180 mm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A dose formulation analysis was conducted for the test item by using validated GC method. The stability, homogeneity, and dose concentration were analysed.

Results:
- stability: dose formulations in the range 1-500 mg/mL have shown to be stable for 7 days when stored at room temperature.
- homogeneity: dose formulations in the range 1-500 mg/mL have shown to be homogeneous.
Homogeneity of dose formulations for dosing start date was determined prior to dosing. Samples were taken from the top, middle and bottom of all dose formulations. Results of homogeneity were 1.92, 0.66 and 1.51% at each dose levels of 30, 100 and 300 mg/kg bw/day. They were acceptable as the Coefficient Variation (CV) was within 10% of the mean of the top, middle and bottom results.
- dose level concentration: results of dose formulations were 106.79, 99.57 and 100.90% at the dose levels of 30, 100 and 300 mg/kg bw/day, respectively. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
Duration of treatment / exposure:
- males: 14 days prior to mating and continued through the day prior to sacrifice (at least 50 days)
- females: 14 days prior to mating and continued through lactation day 13 (exception: non-mated females were sacrificed at least 24 days from final mating day)
Frequency of treatment:
once a day, 7 days/week
Details on study schedule:
not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males / 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose level was selected based on a previous repeated dose 4-week toxicity study (please refer to Section 7.5.1 Repeated dose toxicity: oral: key_Repeated dose toxicity: oral_1994_RL2) in rats resulting in a NOAEL at 150 mg/kg bw/day. Based on the results of this previous study, dose levels of 30, 100 and 300 mg/kg bw/day were selected for this study.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
mortality/morbidity: twice daily, except during acclimatisation; once on the day of necropsy
general clinical signs: once a day during acclimatisation, pre-treatment, and at terminal sacrifice as well as twice a day (before and after dosing) during treatment, mating, post-mating, gestation and lactation (females were additionally observed after parturition was finished)
- Cage side observations checked: mortality and general clinical signs
During the gestation period, dams were especially monitored for signs of abortion or premature delivery. In addition, nursing dams were observed during the lactation period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
acclimation: animal receipt day and day 6 of acclimation
pre-treatment, mating, and post-mating: once per week
treatment: first day of dosing and thereafter once per week
gestation: gestation days 0, 7, 14 and 20
lactation: lactation days 0, 4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule:
treatment and post-mating: once per week
gestation: gestation days 0, 7, 14 and 20
- Amount of food given to each animal and amount of residual feed was measured. Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented. The quantity of food consumption between intervals was presented as g/animal/day.

WATER CONSUMPTION AND COMPOUND INTAKE: No

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- How many animals: all adult males
- Parameters checked: thyroid hormone (T4) concentration

PARTURITION MONITORING:
- mating-proven females were monitored twice daily for signs of parturition including abortion, premature delivery and difficult or prolonged parturition from gestation day 21.
- duration of gestation was recorded
Oestrous cyclicity (parental animals):
- during the pre-treatment period, a vaginal smear was taken daily for each female and regularity and length of the oestrus cycle was examined. Females that fail to exhibit normalestrus cycle were excluded in the study.
- vaginal smear was taken daily from each female from the beginning of the 14 days prior to mating up to the mating period until there was evidence of mating.
- regularity and length of the oestrus cycle during the treatment period until mating was examined.
- vaginal smear of sacrificed females was taken at termination to examine the stage of the oestrus cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
- testis weight (left and right)
- epididymis weight (left and right)
- prostate and seminal vesicles (with coagulation gland)
Organ/body weight ratios using the terminal body weight obtained prior to necropsy were calculated. Paired reproductive organs were weighed separately.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 8 pups/litter (4/sex/litter, if possible); excess pups were killed and discarded.
- litters with 8 or fewer pups were not culled.
- external examinations were conducted on culled pups and subsequently sacrificed.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number of dead and live pups
- runts were recorded
- body weight and sex were recorded on post-natal day 0, 4 (before culling) and 13 (reported as litter)
- external abnormalities were recorded
- mortality, morbidity, and general clinical signs (incl. general appearance and behaviour changes) were recorded for all pups once every day
- anogenital distance was measured in all pups on post-natal day 4. Pup body weight and anogenital distance were collected on the same day and the anogenital distance was
normalized based on the cube root of body weight.
- number of nipples in all male pups was counted on post-natal day 12.

GROSS EXAMINATION OF DEAD PUPS:
yes, external and visceral examinations of dead pups at parturition

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: at post-natal day 13
- How many animals: at least two pups (1 male / 1 female, if possible) per litter
- Parameters checked: thyroid hormone (T4) concentration
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals were euthanized on the day after final dosing.
- Maternal animals: all surviving animals were euthanized on lactation day 14.
Non-mated female was euthanized after at least 24 days from final mating day, and macroscopic findings, tissue preservation and microscopic findings were conducted.
Non-parturition female was euthanized after gestation day 27, and macroscopic findings, tissue preservation, pregnancy confirmation and microscopic findings were conducted.
- all animals were fasted more than 16 hours (overnight) prior to scheduled sacrifice.

GROSS NECROPSY
- complete gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal cavities.
- organs were removed and examined.
- special attention was paid to the organs of the reproductive system.
- lactating dams: number of implantation sites (right and left) was counted
- non-parturition females: uterus implantation sites were examined after staining with ammonium sulfide.
- pregnancy was confirmed by implantation sites in the uterus at sacrifice or parturition.

HISTOPATHOLOGY
Testes, epididymides and ovaries collected from animals at terminal sacrifice in all groups were examined microscopically.
Postmortem examinations (offspring):
GROSS NECROPSY
- external and visceral examinations were conducted in dead pups from birth to day 4 of lactation, if possible.
- all pups on post-natal day 13 were euthanized and then external examinations for abnormalities were conducted.
Statistics:
Mean values and standard deviations were calculated.
Data was considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data was analyzed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were analyzed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and the significance of inter-group differences between the control and treated groups were assessed using Dunn’s Rank Sum Test.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weight. The litter size was used as the covariate. Litter data was statistically evaluated using the statistical unit as a litter.
Data presented as frequencies was analyzed by χ2-test followed by the Fisher's exact test where necessary.
Reproductive indices:
Mating Index (%) = (No. of males with evidence of mating/No. of males paired) × 100
Mating Index (%) = (No. of females with evidence of mating/No. of females paired) × 100
Fertility Index (%) = (No. of males impregnating a female/No. of males paired) × 100
Fertility Index (%) = (No. of pregnant females/No. of females paired) × 100
Fecundity Index (%) = (No. of males impregnating a female/No. of males with evidence of mating) × 100
Pregnancy Index (%) = (No. of pregnant females/No. of females with evidence of mating) × 100
Delivery Index = (No. of dams with live pups/No. of pregnant dams) x 100
Precoital Time = No. of days taken to mate
Offspring viability indices:
Viability Index = (No. of live pups on Day 4 of lactation/No. of live pups at birth) x 100
Weaning Index = (No. of live pups on Day 13 of lactation/No. of pups on Day 4 of lactation after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day: females had a statistically significant increase in body weight gain during the study period (1.13-fold of control). It was considered test itemrelated but not toxicologically significant since there were no correlated changes in other parameters as well as food consumption
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
a statistically significant change in food consumption during the study was not considered test item-related since it was transient.
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:
effects observed, non-treatment-related
Description (incidence and severity):
no test item related changes were observed in the thyroid hormon concentration of adult males
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
changes observed in microscopic observation were considered incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among controls and treated groups.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in thyroid hormone (T4) analysis were observed during the study.
A statistically significant increase of thyroid hormone (T4) in pups at 100 mg/kg was not considered test-item related since it did not have a dose-response.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS:
- 100 and 300 mg/kg bw/day: salivation was observed in both sexes (considered test item-related but not toxicologically significant; attributed to the palatability of the test item).
- other clinical signs were observed but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not have a dose-response.

MORTALITY:
- no deaths or moribund occurred in both sexes of all groups throughout the study.
- two females were sacrifice unschedually. Non-parturition female (control group) was sacrificed on gestation day 28, and non-mated female (30 mg/kg bw/day dose group) was sacrificed after 24 days from final mating day. There were no test item-related changes in macroscopic and microscopic findings.

BODY WEIGHT AND WEIGHT CHANGES.
- no test item-related changes in body weight were observed in both sexes during the study.
- 300 mg/kg bw/day: females had a statistically significant increase in body weight gain during the study period (1.13-fold of control). It was considered test itemrelated but not toxicologically significant since there were no correlated changes in other parameters as well as food consumption.

FOOD CONSUMPTION.
- no test item-related changes in food consumption were observed in both sexes during the study.
- a statistically significant change in food consumption during the study was not considered test item-related since it was transient.

GROSS PATHOLOGY
- no test item-related changes in macroscopic findings were observed in both sexes during the study.
- 30 mg/kg bw/day: one female with cyst of left ovary was considered to be an incidental or spontaneous finding given their low incidence and did not have a dose-response.

HISTOPATHOLOGY
- no test item-related changes in microscopic findings were observed in both sexes during the study.
- changes observed in microscopic observation were considered incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among controls and treated groups.

REPRODUCTIVE FUNCTION. OESTRUS FUNCTION
- no test item-related changes in oestrus cycle were observed during the study.

REPRODUCTIVE FUNCTION: SPERM MEASURES
No test item-related changes in organ weights (testis weight, epididymis weight, and prostate and seminal vesicles (with coagulation gland) were observed during the study.

REPRODUCTIVE PERFORMANCE:
- no test item-related changes in precoital time were observed during the study.
- no test item-related changes in fertility data were observed in both sexes during the study.
- no test item-related changes in reproductive findings were observed during the study.

CLINICAL BIOCHEMISTRY
- no test item related changes were observed in the thyroid hormon concentration of adult males.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One pup with swollen of right eye at 100 mg/kg was considered to be incidental or spontaneous given their low incidence and did not have a dose-response
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One pup with subcutaneous edema of right eye at 100 mg/kg was considered to be incidental or spontaneous given their low incidence and did not have a dose-response.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

- no test item-related changes in littering findings were observed during the study.

CLINICAL SIGNS:
- no test item-related clinical signs were observed in both sexes during the study.
- 100 mg/kg bw/day: one pup with swollen of right eye was considered to be an incidental or spontaneous finding given their low incidence and no dose-response.

BODY WEIGHT AND WEIGHT CHANGES:
- no test item-related body weight changes iwere observed in both sexes during the study.

GROSS PATHOLOGY:
- no test item-related changes in dead pups or at external and visceral examinations.
- no test item-related changes were observed in both sexes of the F1 culled pups during the external examination. One culled pup with subcutaneous oedema of right eye at 100 mg/kg bw/day was considered to be an incidental or spontaneous finding given their low incidence and did not have a dose-response.
- no test item-related changes were observed in both sexes of the non-culled surviving pups during external examination

ANOGENITAL DISTANCE/NIPPLE RETENTION:
- no test item-related changes in anogenital distance were observed in both sexes during the study.
- no test item-related changes in nipple retention of male pups were observed during the study.

CLINICAL BIOCHEMISTRY:
- no test item-related changes in thyroid hormone (T4) analysis were observed during the study.
- 100 mg/kg bw/day: a statistically significant increase of thyroid hormone (T4) in pups was not considered test-item related since it did not have a dose-response.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (general toxicity; rat): 300 mg/kg bw/day (actual ingested)
NOAEL (reproduction/developmental toxicity; rat): 300 mg/kg bw/day (actual ingested)

After the oral administration of 30, 100, and 300 mg/kg bw/day of the test item to male and female adult rats, no adverse test item-related effects were observed for clinical signs, mortality, body weight, body weight gain, food consumption, gross pathology, and histopathology. In addition, no test item-related changes were observed for reproductive function and reproductive performance. No test item related changes were observed in the thyroid hormone concentration of adult males. Lastly, no adverse test item-related findings were made in the F1 pup generation, when investigating clinical signs, mortality, body weight, sexual maturation, thyroid hormone concentration, and gross pathology.
Under the experimental conditions of this screening study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproduction/developmental toxicitywas considered to be 300 mg/kg bw/day (unbound).
Executive summary:

A toxicity to reproduction study was performed with the test item according to the OECD guideline 421 (2016). The test material was suspended in the vehicle (corn oil) and administered by gavage to three groups, each of twelve male and twelve female Crl: CD(SD) rats, at the dose levels of 30, 100, and 300 mg/kg bw/day. The test item was administered once daily to males and females during two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day 13 in females. A concurrent control group received the vehicle only. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, macroscopic findings, organ weights (testis, epididymis, prostate and seminal vesicles only) and microscopic findings (testes, epididymis, and ovaries only) were measured and conducted. In addition, reproductive/developmental observations including oestrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were conducted. Thyroid hormone level in blood was also analysed for adult males and pups at sacrifice.

No deaths or moribund animals occurred in any group throughout the study. In general systemic observations, test item-related salivation was observed in both sexes at 100 and 300 mg/kg, however, it was not considered to have toxicological significance since it was considered to be attributed to the palatability of the test item. In addition, test item related increased weight gain (1.13-fold of control) was observed in females at 300 mg/kg, however, it was not considered to have toxicological significance since there were no correlated changes in other parameters including food consumption.

In reproductive/developmental observations, no test item-related change was observed in estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination.

In thyroid hormone (T4) analysis, no test item-related change was observed in adult males and pups at sacrifice.

Under the experimental conditions of this screening study, the No Observed Adverse Effect Levels (NOAELs) for systemic toxicity and reproduction/developmental toxicity were considered to be 300 mg/kg bw/day.