[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 Aug - 24 Nov 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 Aug - 24 Nov 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

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: 318.0-364.1 g (males), 195.4-239.1 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): 20.4 - 23.1
- Humidity (%): 46.2 - 59.0
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
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 (4.4–6.0°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.
- Amount of vehicle: 5 mL/kg bw

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. As a result of homogeneity and stability analyses conducted the 0.2 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. The results of dose concentration analyses were determined to be 89.85 – 110.76%. These results were within the acceptable limits (± 15% of nominal values).

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

Recovery groups:
Males and females of recovery groups were dosed once daily for 6 weeks. Animals were not mated and were assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

800 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, a decreasing tendency of body weight gain was noted in males and an increase in the absolute and relative liver weights was noted in females at 1000 mg/kg bw/day. Therefore, the high dose level was selected at 800 mg/kg bw/day. Then, the mid and low dose levels were selected at 50 and 200 mg/kg bw/day, respectively.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily - All animals were observed for general condition and clinical signs at least once daily throughout the study. Females were also observed for signs of abortion and pre-mature birth.
- Cage side observations included: mortality/viability

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), leukocyte count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), globulin (Glo), A/G ratio, glucose (Glu), total cholesterol (T-Chol), Triglyceride (TG), sodium (Na), potassium (K), chloride (Cl), calcium (Ca)

URINALYSIS: Yes
- Time schedule for collection of urine: 6 males and females were randomly selected from the main groups in addition to all recovery animals for urinalysis two days before necropsy. Fresh, 3-hour and 24-hour urine samples were collected from the selected animals and analyzed.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters examined: in fresh urine samples: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected animals were examined a few days before necropsy.
- Dose groups that were examined: 6 males and females were randomly selected from the main study groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test, rotarod test, spontaneous motor activity test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All males of the main group were sacrificed 2 weeks after mating and females of the main group were sacrificed on Day 6 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Non-pregnant females were sacrificed on Day 27 after the last day of mating. Complete gross post-mortem examinations were conducted on all animals including the external and internal surfaces. All grossly visible abnormalities were recorded.

ORGAN WEIGHTS: Yes
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: Yes
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 (liver)

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).

Clinical signs:

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
One pregnant female at 0 mg/kg bw/day was found dead on gestation day 22. One female at 50 mg/kg bw/day was found dead on postpartum day (PPD) 2. One female was found dead on PPD 1 at 800 mg/kg bw/day and one on PPD 6 at 200 mg/kg bw/day.
Before females were found dead, clinical signs such as nasal hemorrhage, soiled perineal region, dirty nose and/or staining around mouth were observed.
All males of the main group and all animals of the recovery group survived the duration of the study. In the main group, salivation was sporadically observed in 4 females at 800 mg/kg bw/day from Day 16. In the recovery group, salivation was sporadically observed in 1 male on Day 23 and in 4 females at 800 mg/kg bw/day from Day 24 to Day 40. Salivation was considered to be a test substance-related temporary change since it was observed sporadically and there were no changes on the salivary glands at necropsy and histopathological examination.

BODY WEIGHT AND WEIGHT GAIN
In the main group, no statistically significant differences in body weight changes were noted in males and females compared to the control group.
In males of the recovery group, body weights at 800 mg/kg bw/day were slightly lower than controls from Day 8 to Day 56. In females of the recovery group, body weights at 800 mg/kg bw/day were slightly lower than controls from Day 35 to Day 56. However, it was not considered to be of toxicological significance since these were differences of small magnitude and no significant changes were noted in the main group.

FOOD CONSUMPTION
In the main group, a slight increase in food consumption in males and females at 800 mg/kg bw/day was noted temporarily. However, these changes were not considered to be test substance-related changes since they were not related to body weight changes. There were no significant changes in food consumptions in males and females of the recovery group.

HAEMATOLOGY
In the main and recovery groups, no effects were noted in any animal in any dosing group. Other statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and they were within the range of historical reference data.

CLINICAL CHEMISTRY
In the main and recovery groups, no adverse effects were noted in any animal in any dosing group. Other statistical significances were not considered to be test substance-related changes since these were differences of small magnitude and/or they were within the range of historical reference data.

URINALYSIS
In the main and recovery groups, no effects were noted in any dosing group.

NEUROBEHAVIOUR
In the main and recovery groups, there were no test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test in both sexes when compared to the control group. In the main group, there were no test substance-related effects in the grip strength test and spontaneous motor activity in both sexes when compared to the control group. In the main group, a significant decrease in rotaroad test was noted in females at 800 mg/kg bw/day when compared to control group. However, its value was within the normal ranges of the recovery group. Therefore, this finding was not considered to be of toxicological significance. In the recovery group, statistically significant decreases in grip strength test and spontaneous motor activity were noted in males and/or females at 800 mg/kg bw/day. However, these were not considered to be of toxicological significance since these were differences of small magnitude.

ORGAN WEIGHTS
In the main group, increases in the absolute and/or relative organ weights of the liver were noted in both sexes at 800 mg/kg bw/day. No significant findings were noted for the organ weights in the both sexes of recovery group. However, the increase in the liver organ weight was not considered to be a test substance-related adverse effect since it was not accompanied by increases of ALT and AST and it was reversible in organ weights in the recovery group. Other statistical significances in the absolute and/or relative organ weights were not considered to be test substance-related effects since the differences were small in magnitude and they were within the historical range limit.

GROSS PATHOLOGY
Dead animals:
One female at 0 mg/kg bw/day was found dead before parturition, showing 12 fetuses in the uterus. This animal showed hydrothorax. One female at 50 mg/kg bw/day showed blood clot remained in the left uterine horn. One female at 200 mg/kg bw/day showed discoloration of the heart, entire lobes of the lung and small thymus. One female at 800 mg/kg bw/day showed four placenta and one dead fetus in the uterus, so this animal died because of stillbirth/dystocia. Stillbirth/dystocia is a low incidental finding in the reproductive studies. Therefore, this death was considered to be of no toxicological significance. The clear cause of death could not be determined limited to gross observations. However, the deaths were unrelated to the treatment based on a lack of dose relationship as well as its isolated incidence.
Surviving animals:
Macroscopic examination at necropsy did not reveal test substance-related changes. All other macroscopic findings observed in this study were considered to be incidental and not related to the test substance.

HISTOPATHOLOGY
Test substance-related microscopic findings were not evident in dead animals.
Following the dosing period, test substance-related microscopic findings were present in the liver of surviving animals: hepatocellular hypertrophy was observed in males and females at 800 mg/kg bw/day after 6 weeks of treatment. Hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was in the centrilobular zone. At the end of the 2-week recovery period, this finding disappeared in both sexes at 800 mg/kg bw/day. It was considered to have little toxicological significance since the hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis, and it was completely recovered after a recovery period in this study. No test substance-related histopathological findings were noted in the reproductive organs of either sex. All other microscopic findings seen in various organs and tissues were considered to be incidental and of no toxicological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to and including the highest dose

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was set at 800 mg/kg bw/day, the highest dose tested.

Executive summary:

Males of the main group were dosed once daily for a total of six weeks (two weeks each prior to, during and post mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for five days after delivery. Also, males and females of the recovery groups were dosed for six weeks.

All males of the main group and all animals of recovery group survived the duration of the study. In the main group, each one female died at 0, 50, 200 and 800 mg/kg/day. However, the deaths were unrelated to the treatment based on a lack of dose relationship as well as its isolated incidence.

No test substance-related adverse effects were noted in the results of detailed clinical signs, body weights, food consumption, sensory function, motor activity, urinalysis, hematology and blood biochemistry in animals of the test substance-treated groups.

In the main group, increases in the absolute and/or relative organ weights of the liver were noted in both sexes at 800 mg/kg/day. Hepatocellular hypertrophy was observed in both sexes at 800 mg/kg/day. It was considered to have little toxicological significance since the hepatocellular hypertrophy in centrilobular zone is generally considered to be an adaptive response in nature.

There were no test substance-related effects on the mating period, mating index, gestation period, male and female fertility indexes, gestation index, pre- and post-implantation loss rates, live birth index, mean litter size, external examination of pups, body weights of pups and sex ratio of pups and viability index of postnatal Days 0 and 4.

Based on these results of this study, the NOAEL of Menthyl methyl ether for systemic and reproductive toxicity studies was considered to be 800 mg/kg/day for males and females. The NOAEL for pups was considered to be 800 mg/kg/day.

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:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

EC Number:

216-365-6

EC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Cas Number:

1565-76-0

Molecular formula:

C11H22O

IUPAC Name:

[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

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: 318.0-364.1 g (males), 195.4-239.1 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): 20.4 - 23.1
- Humidity (%): 46.2 - 59.0
- 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 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 (4.4–6.0 °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.
- Amount of vehicle: 5 mL/kg bw

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. As a result of homogeneity and stability analyses conducted the 0.2 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. The results of dose concentration analyses were determined to be 89.85 – 110.76%. These results were within the acceptable limits (± 15% of nominal values).

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

Recovery groups:
Males and females of recovery groups were dosed once daily for 6 weeks. Animals were not mated and were 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 / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

800 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, a decreasing tendency of body weight gain was noted in males and an increase in the absolute and relative liver weights was noted in females at 1000 mg/kg bw/day. Therefore, the high dose level was selected at 800 mg/kg bw/day. Then, the mid and low dose levels were selected at 50 and 200 mg/kg bw/day, respectively.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed for general condition and clinical signs at least once daily throughout the study. Females were also observed for signs of abortion and pre-mature birth. 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 postpartum 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:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
One pregnant female at 0 mg/kg bw/day was found dead on gestation day 22. One female at 50 mg/kg bw/day was found dead on postpartum day (PPD) 2. One female was found dead on PPD 1 at 800 mg/kg bw/day and one on PPD 6 at 200 mg/kg bw/day.
Before females were found dead, clinical signs such as nasal hemorrhage, soiled perineal region, dirty nose and/or staining around mouth were observed.
All males of the main group and all animals of the recovery group survived the duration of the study. In the main group, salivation was sporadically observed in 4 females at 800 mg/kg bw/day from Day 16. In the recovery group, salivation was sporadically observed in 1 male on Day 23 and in 4 females at 800 mg/kg bw/day from Day 24 to Day 40. Salivation was considered to be a test substance-related temporary change since it was observed sporadically and there were no changes on the salivary glands at necropsy and histopathological examination.

BODY WEIGHT AND WEIGHT GAIN
In the main group, no statistically significant differences in body weight changes were noted in males and females compared to the control group.
In males of the recovery group, body weights at 800 mg/kg bw/day were slightly lower than controls from Day 8 to Day 56. In females of the recovery group, body weights at 800 mg/kg bw/day were slightly lower than controls from Day 35 to Day 56. However, it was not considered to be of toxicological significance since these were differences of small magnitude and no significant changes were noted in the main group.

FOOD CONSUMPTION
In the main group, a slight increase in food consumption in males and females at 800 mg/kg bw/day was noted temporarily. However, these changes were not considered to be test substance-related changes since they were not related to body weight changes. There were no significant changes in food consumptions in males and females of the recovery group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating indices of the groups at 0, 50, 200 and 800 mg/kg bw/day were all 100.0%. Male and female fertility indexes of these groups were 91.7, 91.7, 91.7 and 100.0%, respectively. While the mating period of the groups was 2.8, 2.2, 2.5 and 1.9, the gestation period was 22.4, 22.2, 22.0 and 21.9, respectively. There were no significant differences in mating, fertility index and mating period in any dose group.
The gestation period of the groups at 200 and 800 mg/kg bw/day was significantly shorter than the control group. However, these changes were not considered to be test substance-related changes since there were normal gestation period and within historical reference data.
Pre-implantation loss rates of the groups at 0, 50, 200 and 800 mg/kg bw/day were 12.1, 10.4, 9.0 and 13.2% and post-implantation rates of these groups were 8.7, 15.9, 8.9 and 10.8%, respectively. The mean litter sizes were 14.5, 14.2, 15.5 and 14.9, respectively.
Normal delivery was observed in females at 50 and 200 mg/kg bw/day. Stillbirth/dystocia was observed in one dam at 800 mg/kg bw/day. Stillbirth/dystocia is a low incidental finding in the reproductive studies. Therefore, dystocia was considered to be of little toxicological significance.

ORGAN WEIGHTS
In the main group, increases in the absolute and/or relative organ weights of the liver were noted in both sexes at 800 mg/kg bw/day. No significant findings were noted for the organ weights in the both sexes of recovery group. However, the increase in the liver organ weight was not considered to be a test substance-related adverse effect since it was not accompanied by increases of ALT and AST and it was reversible in organ weights in the recovery group. Other statistical significances in the absolute and/or relative organ weights were not considered to be test substance-related effects since the differences were small in magnitude and they were within the historical range limit.

GROSS PATHOLOGY
Dead animals:
One female at 0 mg/kg bw/day was found dead before parturition, showing 12 fetuses in the uterus. This animal showed hydrothorax. One female at 50 mg/kg bw/day showed blood clot remained in the left uterine horn. One female at 200 mg/kg bw/day showed discoloration of the heart, entire lobes of the lung and small thymus. One female at 800 mg/kg bw/day showed four placenta and one dead fetus in the uterus, so this animal died because of stillbirth/dystocia. Stillbirth/dystocia is a low incidental finding in the reproductive studies. Therefore, this death was considered to be of no toxicological significance. The clear cause of death could not be determined limited to gross observations. However, the deaths were unrelated to the treatment based on a lack of dose relationship as well as its isolated incidence.
Surviving animals:
Macroscopic examination at necropsy did not reveal test substance-related changes. All other macroscopic findings observed in this study were considered to be incidental and not related to the test substance.

HISTOPATHOLOGY
Test substance-related microscopic findings were not evident in dead animals.
Following the dosing period, test substance-related microscopic findings were present in the liver of surviving animals: hepatocellular hypertrophy was observed in males and females at 800 mg/kg bw/day after 6 weeks of treatment. Hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was in the centrilobular zone. At the end of the 2-week recovery period, this finding disappeared in both sexes at 800 mg/kg bw/day. It was considered to have little toxicological significance since the hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis, and it was completely recovered after a recovery period in this study. No test substance-related histopathological findings were noted in the reproductive organs of either sex. All other microscopic findings seen in various organs and tissues were considered to be incidental and of no toxicological significance.

OTHER:
For results on haematology, clinical chemistry, urinalysis, neurobehavioural examination refer to IUCLID section 7.5.1.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects and no adverse effects on reproduction up to and including the highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

only external

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Live birth indices of the groups at 0, 50, 200 and 800 mg/kg bw/day were 91.3, 84.1, 91.1 and 89.2%, respectively. In these groups, viability indices on postnatal Day (PND) 0 were 94.2, 95.6, 98.9 and 92.3, and the indices on PND 4 were 87.7, 97.7, 89.4 and 94.9%, respectively.

BODY WEIGHT (OFFSPRING)
There were no treatment-related changes in body weights of pups at 50, 200 and 800 mg/kg bw/day.

GROSS PATHOLOGY (OFFSPRING)
There were no test substance-related changes in external findings of pups at 50, 200 and 800 mg/kg bw/day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 800 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)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for systemic and reproductive toxicity was set at 800 mg/kg bw/day, the highest dose tested. No adverse effects on development of offspring were observed up to and including 800 mg/kg bw/day, the highest dose tested.

Executive summary:

There were no test substance-related effects on the mating period, mating index, gestation period, male and female fertility indexes, gestation index, pre- and post-implantation loss rates, live birth index, mean litter size, external examination of pups, body weights of pups and sex ratio of pups and viability index of postnatal Days 0 and 4.

Based on these results of this study, the NOAEL of Menthyl methyl ether for reproductive toxicity studies was considered to be 800 mg/kg/day for males and females. The NOAEL for pups was considered to be 800 mg/kg/day.