Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct 2015 - 5 Feb 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22 Mar 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate
EC Number:
946-282-7
Molecular formula:
C11H20O2
IUPAC Name:
reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

Test animals

Species:
rat
Strain:
other: Crl:CD(SD), SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks (male), 8 weeks (female)
- Weight at study initiation: 312.4 - 349.0 g (males), 189.0 - 226.4 g (females)
- Housing: acclimation period and pre-mating: 1 animal per cage; mating: 1:1; lactation: neonates were kept with the dam; animals were kept in stainless wire mesh cages (260W x 350D x 210H mm) and in polycarbonate cages (260W x 420L x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Harlan Laboratories, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 23.9
- Humidity (%): 47.0 - 57.5
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer and then, the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (5.7 – 7.6 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was dissolved in it.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: All females were examined for the presence of a vaginal plug or sperm in the vaginal smear twice a day for confirmation of mating. Positive confirmation was defined as Day 0 of Gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be 101.64 - 103.74%. These results were within the acceptable limits (± 15% of nominal values).
As a result of homogeneity and stability analyses conducted the 0.5 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 h at room temperature and for 7 days under refrigeration.
Duration of treatment / exposure:
Main groups:
males: for 6 weeks, starting 2 weeks before mating, during mating and 2 weeks after mating
females: for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy; females showing no evidence of mating were dosed daily for 26 days after the last day of mating

Recovery groups:
males and females: once daily for 6 weeks; animals were not mated and assigned to 2 weeks of recovery period after the completion of administration
Frequency of treatment:
once daily, 7 days/week
Details on study schedule:
not applicable for an OECD 422 study
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 (main groups)
6 (recovery groups; for control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 2-week repeated oral dose range-finding study, an increase in organ weights of the kidney and liver and bilateral enlargement of the kidney were observed in males at 1000 mg/kg bw/day. Therefore, the high dose level was selected at 500 mg/kg bw/day. Then, the low and mid dose levels were selected at 50 and 150 mg/kg bw/day, respectively.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: general condition and clinical signs, mortality/viability, abortion and pre-mature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations for signs and symptoms of adverse effects, including central and autonomic nervous system effects, motor activity and behavior, were conducted on all animals once before the test and once a week throughout the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day before necropsy and on the day of necropsy (fasted body weights). Body weights of females of the main group were recorded just prior to dosing on Day 1, once a week throughout the dosing and recovery periods, on Days 0, 7, 14 and 20 of gestation, on Days 0 and 4 post partum and on the day of necropsy (fasted body weights). Fasted body weights recorded on the day of necropsy were presented, but were not included in statistical analysis.

FOOD CONSUMPTION: Yes
- Food consumptions of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1, once a week during the dosing and recovery periods and the day before necropsy. Food consumptions of females of the main group were recorded just prior to dosing on Day 0, once a week throughout the dosing and recovery periods, on Days 0, 6, 13 and 19 of gestation, on Days 0 and 3 post partum. Food consumption was not recorded during mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OTHER: haematology, clinical chemistry, urinalysis, neurobehavioural examination (for details refer to IUCLID section 7.5.1)
Sperm parameters (parental animals):
Histopathological examination was conducted with focus on spermatogenesis.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, body weight, presence of gross abnormalities, postnatal mortality
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals of the main group were sacrificed 2 weeks after mating.
- Maternal animals: All animals of the main group were sacrified on Day 6 postpartum. Non-pregnant females were sacrificed on Day 27 after the last day of dosing.
All animals of the recovery group were sacrified 2 weeks after final dosing.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All grossly visible abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. The testes and epididymides of all adult males were weighed. 6 males and females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed: brain, heart, liver, thymus, spleen, kidneys, adrenals, ovaries, uterus

Histopathology
Tissue preservation and slide preservation
6 males and females were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides were fixed in Bouin's solution. The eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10% neutral buffered formalin

For the histopathological examination, the preparation of specimens of organs and tissues was carried out and the remaining organs and tissues preserved in 10% neutral buffered formalin: brain, pituitary, thymus, lung with bronchi, trachea, thyroid, esophagus, heart, liver, spleen, kidneys, adrenals, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymides, prostate, ovaries, uterus, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye and optic nerve, urinary bladder, gross lesions

Besides, from all animals except for six females and males in the main group, the following organs and tissues were harvested and preserved: brain, pituitary, heart, thymus, liver, spleen, kidneys, adrenals, prostate, testes, epididymides, ovaries, uterus

Histopathological examinations were conducted as follows:
- 6 males and females from the control, low, mid and high dose group (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead or killed in a moribund condition
- All gross, macroscopic lesions
- Target organs noted at the high dose were examined for the recovery group
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS 9.3). For the data including body weights, food consumption, urine volume and specific gravity, hematology and blood biochemistry parameters, organ weights, mating result, birth and survival rates, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant (significance level: 0.05), followed by Dunnett’s t-test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneity, if significant (significance level: 0.05), followed by Steel’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed on homogeneity, if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).
Reproductive indices:
Mating index (%) = (number of females with confirmed mating / number of females placed with males) x 100
Mating period = Day of mating confirmed - Day of initial mating (based on dosing day)
Gestation period = Day 0 post partum - Day 0 of gestation (based on dosing day)
Male fertility index (%) = (number of males impregnating a female / number of males with confirmed mating) x 100
Female fertility index (%) = (number of pregnant females / number of females with confirmed mating) x 100
Gestation index (%) = (number of females with live pups / number of pregnant females) x 100
Pre-implantation loss (%) = [(number of corpus lutea - number of implantations) / number of corpus lutea] x 100
Post-implantation loss (%) = [(number of implantations - number of live pups) / number of implantations] x 100
Offspring viability indices:
Live birth index (%) = (number of live pups on postnatal Day 0 / number of implantations) x 100
Viability index on postnatal Day 0 (%) = (number of pups born alive on postnatal Day 0 / total number of pups born) x 100
Viability index on postnatal Day 4 (%) = (number of pups surviving on postnatal Day 4 / number of pups born alive on postnatal Day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the main groups, soiled perineal region was observed frequently in males and females at 500 mg/kg bw/day from Day 15 to the final dosing day. Also, soiled perineal region was observed once in one male and female at 150 mg/kg bw/day on Day 15. Salivation was observed often in males at 500 mg/kg bw/day from Day 22 and sporadically in females at 500 mg/kg bw/day from Day 6 of gestation. In the recovery groups, soiled perineal region was observed in one male and five females at 500 mg/kg bw/day from Day 21. Salivation was observed in three males and one female at 500 mg/kg bw/day.

No clinical signs were observed in any animal of the control and dosed groups in the detailed examinations once a week.
Mortality:
mortality observed, treatment-related
Description (incidence):
All males of the main group and all animals of the recovery groups survived the duration of the study. Two females of the main group were found dead at 500 mg/kg bw/day on Days 3 and 5 post-partum, respectively. Before females were found dead, clinical signs such as soiled perineal region, staining around mouth and/or hematuria were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences in body weight changes were noted in males of the main group and in animals of both sexes in the recovery groups, but a statistically significantly high value was noted in females at 500 mg/kg bw/day in the main group on Day 0 post-partum (+9.7% vs. control), which was considered to be a test substance-related effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly increase in food consumption (+18.5%) was noted in males at 500 mg/kg bw/day in the main group on Day 42. A statistically significant increase in food consumption was noted in females at 150 and 500 mg/kg bw/day in the main group on Day 7 of gestation (+17.8 and +18.2%, respectively) and at 500 mg/kg bw/day on Day 14 of gestation (+14.4%). A statistically significant increase in food consumption was noted in males at 500 mg/kg bw/day in the recovery group on Days 8, 21 and 36 (+9.4, +12.7 and +27.5%, respectively). A statistically significant decrease in food consumption was noted in females in the recovery group on Day 29 (-8.6%).
A statistically significantly high value in food consumption was noted in females at 500 mg/kg bw/day in the main group during the dosing period. It was considered to be a test substance-related effect since it was associated with increase in body weights.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No effects were observed in any animal in the main and recovery groups. Other statistical significances were considered not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects were observed in any animal in the main and recovery groups. Other statistical significances were considered not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Proteinuria was observed in two males and two females at 500 mg/kg bw/day in the main groups and in one female at 500 mg/kg bw/day in the recovery group. Occult blood and erythrocyte were observed in one female at 150 mg/kg bw/day in the main group. However, there were no changes in the blood biochemistry and histopathological changes related to proteinuria, occult blood and erythrocytes in urine. Therefore, these changes were considered to be of little toxicological significance since no significant changes were observed in the histopathological examination.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main and recovery groups when compared to the control group. In animals of both sexes in the main groups, there were no test substance-related effects in the grip strength test, rotarod test and spontaneous motor activity when compared to the control group. In males of the recovery group, a statistically significant increase in grip strength test was noted at 500 mg/kg bw/day, but it was not considered to be of toxicological significance because of small magnitude.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Marked thymic lymphoid atrophy (2/2) and mild splenic lymphoid atrophy (2/2) were observed in both dead females at 500 mg/kg bw/day in the main group. These lesions correlated with necropsy findings of small thymus and spleen. In addition, mild adrenal cortical hypertrophy (1/2) and mild erosion/ulceration of stomach (1/2) were observed. These findings are frequently observed in rats under poor conditions and are effects related to test substance.
Histopathological examination of surviving animals revealed hepatocellular hypertrophy in animals of both sexes at 500 mg/kg bw/day in the main groups after six weeks of treatment. Hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within the centrilobular zone. At the end of the 2-week recovery period, this finding disappeared in animals of both sexes at 500 mg/kg bw/day. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis. Furthermore, the effect was completely reversible after the recovery period in this study. No test substance-related histopathological findings were noted in the reproductive organs of either sex.
Minimal and mild to moderate accumulation of hyaline droplets in the cortical tubular epithelium of the kidney was observed in all males of the test substance-dosed groups. Hyaline droplets were not observed in animals after the 2-week recovery period. The increased incidence and severity of this lesion were considered to be induced by the test substance. In addition, the affected tubules were not associated with any other visible evidence of tubular injury such as degeneration, inflammation or necrosis. Hyaline droplets are frequently observed in the cytoplasm of proximal tubules in male rats and characterized by variably sized, refractile, brightly eosinophilic droplets consisted of lysosomal accumulation of alpha-2u-globulin. A large number of agents were reported to result in alpha-2u-globulin nepropathy in males only. Therefore, this male-rat-specific effect was not considered to be of human relevance.
All other microscopic findings in various organs and tissues were considered to be incidental and of no toxicological significance.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test substance-related histopathological findings were noted in the reproductive organs.
Reproductive performance:
no effects observed
Description (incidence and severity):
In the control and 50, 150 and 500 mg/kg bw/day dose groups, the mating periods were 2.3, 2.8, 2.8 and 3.7 days, the mating index was 100%, the gestation period was 22.1 days and the fertility indices of animals of both sexes were 100.0, 91.7, 91.7 and 100.0%, respectively. There were no statistically significant differences in any dosed group.
The gestation index of the control and 50, 150 and 500 mg/kg bw/day dose group was 100%, the pre-implantation loss rates were 25.0, 18.8, 18.4 and 20.1%, the post-implantation loss rates were 12.4, 3.5, 6.2 and 12.4% and the mean litter sizes were 14.0, 15.6, 14.4 and 14.1.
In the 50 mg/kg bw/day dose group, a statistically significantly low value of post-implantation loss rate and a statistically significantly high value of live birth index were noted, but these effects were not confirmed as adverse effects.
In addition, normal delivery was observed in all animals of the control and 50, 150 and 500 mg/kg bw/day dose groups, and the sex ratios on PND 0 were 1.2, 1.0, 1.2 and 1.0, respectively.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on reproduction up to and including the highest dose tested

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test substance-related adverse effects were noted in live birth index and in viability index of postnatal Days 0 and 4.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related adverse effects were noted in body weight of pups.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related adverse effects were noted in external examination of pups.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Live birth indices of the groups at 0, 50, 150 and 500 mg/kg bw/day were 87.7, 96.5, 93.8 and 87.6%, respectively. In these groups, viability indices on postnatal Day (PND) 0 were 97.6, 100.0, 99.4 and 98.5%, and the indices on PND 4 were 98.9, 95.1, 98.7 and 89.1%, respectively.

BODY WEIGHT (OFFSPRING)
A statistically significantly high value of body weights was noted in female pups at 500 mg/kg bw/day on PND 4 (+21.3% vs. control), however, it was not considered to be an adverse effect.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related changes in external findings of pups at 50, 150 and 500 mg/kg bw/day.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on development of offspring up to and including the highest dose tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for reproductive toxicity was set at 500 mg/kg bw/day, the highest dose tested. No adverse effects on development of offspring were observed up to and including 500 mg/kg bw/day.
Executive summary:

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2016). Twelve Sprague Dawley rats per sex and dose were treated via gavage with test substance at concentrations of 50, 150 and 500 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, a recovery group of 6 rats per sex was allocated to the control and high dose group. Males were treated for 6 weeks, starting 2 weeks before the mating period, during mating and 2 weeks after mating. Females were treated for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy. Females showing no evidence of mating were dosed daily for 26 days after the last day of mating. The doses were selected on the basis of data from a range-finding study. In parental animals, no effects on reproductive function (spermatogenesis) or performance (male and female mating, fertility, pre- and post-implantation loss rates, gestation index and duration) were observed, compared with the control group. A significantly low value of post-implantation loss rate was noted at 50 mg/kg bw/day. However, this effect was not considered as adverse effects. No test substance-related findings were observed on reproductive organs in males and females at any dose group.

Therefore, a NOAEL for parental fertility of ≥500 mg/kg bw/day was derived for male and female rats.