2,6-Octadienal, 3,7-dimethyl-, acid-isomerized

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.03.-29.09.2017

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.03.-29.09.2017

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:

July 29, 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Number at receipt: 136 (Male 66, Female 70)
- Number on study: 120 (Male 60, Female 60)
- Age range at receipt: Approximately 7 weeks (Males) and 9 weeks (Females)
- Age range on study: Approximately 10 weeks (Males) and 12 weeks (Females)
- Body weight range: 338.7-404.8 g at the initiation of dosing (Males); 238.0-287.7 g at the initiation of dosing (Females)
- Method of identification: Color marking, Tail tattoo, Cage card
- Health status:Healthy animals were used in this study
- Acclimation: All animals were permitted an acclimation period of 6 days to the laboratory environment
- Animal assignment (Randomization): Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and group assignment), estrus cycle and free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.

HOUSING AND ANIMAL CARE:
- Housing: stainless-steel cage (255W×465L×200H mm)
- Number of animals per cage: Acclimation 2 , Pre-treatment 2 , Treatment 2 or 1, Recovery 2 or 1
- Type of Cage: stainless-steel cage (255W×465L×200H mm)
- Temperature: 21.1 - 24.1°C
- Humidity: 40.9 – 57.6%
- Light cycle: 12 hour light/12 hour dark cycle
- Light intensity: 150 - 300 Lux
- Air changes: 10 - 20 times/hour
- Cleaning: Animal room was cleaned daily. Stainless-steel cages were cleaned at least once per 2 weeks, and poly sulfone cages were cleaned at least once per week.

Food, Water and Bedding:
- A standard rat pellet diet was provided to the animals ad libitum. Microbial monitoring for diet was performed
- The animals have had ad libitum access to filtered, ultraviolet light-irradiated municipal tap water at all times. The drinking water was analyzed every 6 months for specified contaminants
- Aspen animal bedding material was sterilized and then provided to the animals in each cage. A certificate of analysis for the bedding was provided by the supplier.
There were no known contaminants in the food, water and bedding material at levels that would be expected to interfere with the results of the study and the data was maintained in the raw data.

Route of administration:

oral: gavage

Details on route of administration:

Dose was based on the most recently measured body weight. Animals were dosed at a volume of 2 mL/kg. Dose formulation was continuously stirred by magnetic stirrer in the animal room (before and throughout the dosing procedure).

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The control item or dose formulations were administered by oral gavage once a day at approximately the same time each day (08:30 - 12:30).
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer about 5 min 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 formulations were transferred to the animal room at room temperature.

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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.

Verification of dose level concentrations:
Analyses of the dosing formulations were conducted and samples for homogeneity analysis were also analyzed for verification of dose level concentration. Results of dose formulations were 107.21, 106.87 and 106.88% at each dose levels of 35, 150 and 600 mg/kg. They were acceptable as the mean concentration was within ±15% of the nominal concentration

Duration of treatment / exposure:

Dosing of the males began 14 days prior to mating and continued throughout mating prior to sacrifice (at least 50 days).
Dosing of the females was beginning 14 days prior to mating and continued throughout lactation day (LD) 13.
Animals of the recovery group were not mated and assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

7 days/week once daily

Dose / conc.:

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

Remarks:

vehicle control (VC)

Dose / conc.:

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

Remarks:

T1

Dose / conc.:

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

Remarks:

T2

Dose / conc.:

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

Remarks:

T3

No. of animals per sex per dose:

12 animals per sex per group
Additional recovery groups: 0 and 600 mg/kg (6 animals per sex per group)

Control animals:

yes, concurrent vehicle

Details on study design:

120 animals (60 males and 60 females) were included in the study (at the start of administration).
All animals were examined for general health upon receipt.
Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and group assignment). They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight. All assigned animals were permitted a pre-treatment period of 14 days before dosing start.

- Dose selection rationale:
The dose level was selected based on the results of a two weeks dose range-finding study.

Positive control:

not applicable

Observations and examinations performed and frequency:

IN-LIFE OBSERVATIONS AND MEASUREMENTS

Mortality observation:
Mortality and morbidity observations were conducted twice daily except for the acclimation period. In addition, it was conducted once on necropsy day.

General clinical signs :
Clinical signs including mortality, moribundity, general appearance, behavior changes were recorded with date/time of finding, and duration.
Animals judged to be abnormal were examined by a veterinarian or an experienced technician.

Detailed clinical signs:
Detailed clinical observations were made in all animals individually for abnormalities. Signs noted were included (but are not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition.
General clinical signs after dosing were not additionally recorded in the day of detailed clinical signs observation.

Body weight (Period/Frequency):
- Acclimation: Animal receipt day and day 6 of acclimation
- Pre-treatment: one time per week
- Treatment: first day of dosing and thereafter one time per week
- Recovery: one time per week
- Necropsy: the day prior to necropsy and on the day of necropsy

Food consumption (Period/Frequency):
- Treatment: one time per week and for females on Gestation Days 0, 6, 13 and 19, on Postpartum Days 0, 3 and 12
- Recovery: one time per week
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.

Functional observations:
Sensory function tests (approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity were conducted in first six animals per sex in main group (if possible) and all animals in recovery group shortly before scheduled sacrifice.

CLINICAL PATHOLOGY
Blood collection:
Blood samples for clinical pathology were taken from all scheduled sacrifice animals in main and recovery groups. Blood samples were collected from abdominal vena cava.

Hematology:
Approximately 1.5 mL of blood samples was collected from all scheduled sacrifice animals in main and recovery groups.
Hematology Parameters:
Total leukocyte count (WBC), Mean corpuscular hemoglobin (MCH), Total red blood cell count (RBC), Mean corpuscular hemoglobin concentration (MCHC), Hemoglobin (HGB), Platelet count (PLT), Hematocrit (HCT), Reticulocyte count, Mean corpuscular volume (MCV), WBC differential count, Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB).


Clinical chemistry:
Approximately 1.5 mL of blood samples was collected from all scheduled sacrifice animals in main and recovery groups and put into tubes without anticoagulant for serum separation.
Clinical Chemistry Parameters:
Glucose (GLU), Alanine aminotransferase (ALT), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CREA), Alkaline phosphatase (ALP), Total protein (TP), Gamma glutamyl transpeptidase (GGT), Albumin (ALB), Creatine phosphokinase (CK), Albumin/globulin ratio (A/G), Calcium (Ca), Total cholesterol (TCHO), Inorganic phosphorus (IP), Triglyceride (TG), Sodium (Na), Phospholipid (PL), Potassium (K), Aspartate aminotransferase (AST), Chloride (Cl).

Thyroid Hormone (T4 and TSH) Analysis:
Blood samples (approximately 0.6 mL) for thyroid hormone (T4 and TSH) analysis was collected.
Thyroid hormone (T4 and TSH) concentration in the serum samples (all adult males in main/recovery groups) were analyzed.

Sacrifice and pathology:

Unscheduled Sacrifice:
During the study period, all unscheduled sacrificed animals were euthanized CO2 at the discretion of a study director and/or an attending veterinarian.

Macroscopic findings:
Complete necropsy was performed under the direct supervision of a veterinarian pathologist. After blood sampling, the animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined.

Tissue preservation:
Tissues from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximate 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder. Tissues from unscheduled sacrificed animals were processed for microscopic evaluation.

Scheduled Sacrifice:
Method of termination
All surviving main group males were euthanized on the day after final dosing using isoflurane.
All surviving main group females on LD 14 were euthanized using isoflurane.
All surviving recovery group males and females were euthanized using isoflurane at least 14 days after the first scheduled sacrifice of males and females.
All animals were fasted more than 16 hours (overnight) prior to scheduled sacrifice.

Macroscopic findings:
Complete necropsy was performed under the direct supervision of a veterinary pathologist. After blood sampling, the animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined.

Organ weights:
Organs were weighed for all animals at terminal and recovery sacrifice groups, and organ/body weight ratios using the terminal body weight (TBW) obtained prior to necropsy were calculated. However, paired reproductive organs were weighed separately.

Tissue preservation:
Tissues from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximate 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.

Microscopic findings:
Tissues except reproductive organs and thyroids (with parathyroids) collected from the first six animals per sex in the main group were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. In addition, all reproductive organs (ovaries, prostate, testes, epididymides, uterus with cervix and seminal vesicles with coagulation gland) and abnormal lesions collected from all animals in the main group were further processed to slides, stained with H&E, and examined microscopically. Thyroids (with parathyroids) for adult males were further processed to slides, stained with hematoxylin and eosin, and examined microscopically to clarify the change of thyroid hormone. Any target organs were additionally evaluated microscopically from recovery group animals.

Statistics:

Mean values and standard deviations were calculated in the final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima System or Statistical Analysis Systems.
Data from non-pregnant females until mating will be included in the report. Data were 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.
For comparing control group and recovery group, the data was analyzed for homogeneity for variance using F-test.
Homogeneous data was analyzed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test.
Heterogeneous data was analyzed using Kruskal-Wallis Test and significant difference between control and recovery group was assessed using Dunn’s Rank Sum Test.
Data presented as frequencies was analyzed by χ2-test followed by the Fisher's exact test where necessary.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In both sexes at ≥ 150 mg/kg, salivation was observed.
Other clinical signs during the study were not considered test item-related since these findings were observed with low frequency, occurred sporadically, generally of low severity and/or did not have a dose-response.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female (Animal No. 115) in the recovery group at 600 mg/kg was found dead on dosing day 47. Macroscopic findings included minimal enlarged adrenal glands and partial irregular surface of the right kiney. Microscopic analysis revealed marked pyelonephritis in the kidneys, severe necrosis in the adrenal glands and slight hepatocyte hypertrophy in the liver.
Unscheduled sacrifice was performed on eight females (Animal No. 103, 105, 106, 108, 110, 112, 113 and 114) at 600 mg/kg .
In the results of microscopic findings, minimal to slight hepatocyte hypertrophy in the liver was observed in some unscheduled sacrificed animals, but there were no changes in reproductive organs

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 600 mg/kg, decreased body weight gain in the main group (89% of control) and recovery group (79% of control) during the treatment period was observed.
In females at 600 mg/kg, decreased body weight gain in the main group (85% of control) and recovery group (58% of control) during the treatment period was observed.
Other statistically significant change in body weight was not considered test-item related since it was transient.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In females at 600 mg/kg, decreased food consumption in the main group (89% of control) during the treatment period was observed.
Other statistically significant changes in food consumption were not considered test-item related since it was minimal or transient.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
In males at 600 mg/kg, decreased total red blood cell count (RBC, 92% of control), hemoglobin (HGB, 92% of control), and hematocrit (HCT, 91% of control) were observed.
Other statistically significant changes in hematology were not considered test item-related since it was minimal or only observed in the recovery group.

Coagulation:
In males at ≥ 150 mg/kg, increased prothrombin time (PT, up to 1.13-fold over control) and at 600 mg/kg decreased fibrinogen (FIB, 95% of control) were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males at 600 mg/kg, increased blood urea nitrogen (BUN, 1.17-fold over control), albumin/globulin ratio (A/G, 1.07-fold over control) and gamma glutamyl transpeptidase (GGT, 1.68-fold over control) and decreased triglyceride (TG, 41% of control) were observed. In females at 600 mg/kg, increased GGT (2.21-fold over control) was observed.
Other statistically significant changes in clinical chemistry were not considered test item-related because there no dose-relationship, no correlated microscopic changes and/or observed only in recovery group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related changes in functional behavior examination were observed in both sexes during the study.

Motor activity examination:
No test item-related changes in motor activity examination were observed in both sexes during the study.
A statistically significant change in motor activity examination was not considered test item-related since it was transient and did not have a dose-response.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males at 600 mg/kg, increased absolute and relative weights of kidneys (absolute weights: 1.25-fold over control, relative weights: 1.29-fold over control) and liver (absolute weights: 1.45-fold over control, relative weights: 1.51-fold over control) were observed. At 150 mg/kg, increased relative weight of liver (1.11-fold over control) was observed.
In females at ≥ 35 mg/kg, increased relative weight of liver (up to 1.48-fold over control) was observed. At ≥ 150 mg/kg, increased absolute weight of liver (1.33-fold over control) was observed.
Other statistically significant changes in organ weights were not considered test item-related, because there were no dose-relationship, no correlated microscopic changes and/or observed only in recovery group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in macroscopic findings were observed in both sexes during the study.
Macroscopic findings observed during this study were considered incidental or spontaneous and are commonly seen in this animal strain given their low incidence or severity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In both sexes at 600 mg/kg, minimal to slight hepatocyte hypertrophy in liver was observed. In males at 600 mg/kg, minimal to slight follicular epithelial hypertrophy in thyroid glands was observed. In addition, minimal to slight tubular basophilia in kidney and minimal to slight ductal atrophy in epididymides were observed.
Other changes observed in microscopic findings were considered incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among control and treated groups.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone (T4 and TSH) Analysis:
In males at 600 mg/kg, decreased T4 (73% of control) was observed.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general systemic effects

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Conclusions:

The No-Observed-Adverse-Effect Level (NOAEL) for general systemic toxicity when the test item is administered via oral gavage to Sprague-Dawley rats with dose levels of 0, 35, 150 and 600 mg/kg bw/day is considered to be 150 mg/kg/day for the animals of both sexes.

Executive summary:

The objective of this study was to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic toxicity after repeated dose oral gavage administration in Sprague-Dawley rats. The assay was performed in accordance to OECD TG 422 and in compliance to GLP.

This study was conducted to evaluate the potential toxicities of the test item regarding general systemic effects. The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 35, 150 and 600 mg/kg with a dose volume of 2 mL/kg. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (total 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in the recovery groups 0 and 600 mg/kg (6 animals per sex per group) received the test item. Afterwards, they were assigned to 2 weeks of recovery period after the completion of test item administration. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and evaluated. Thyroid hormone (T4 and TSH) level in blood was also analyzed for adult males at sacrifice.

One female in recovery group at 600 mg/kg was found dead on dosing day 47. Microscopic findings including marked pyelonephritis in the kidneys, severe necrosis in the adrenal glands, and slight hepatocyte hypertrophy in the liver were observed. The cause of death was considered renal pyelonephritis but not considered test item-related.

Six non-parturition females and 2 non-mated females performed unscheduled sacrifice. Microscopic findings including minimal to slight hepatocyte hypertrophy in the liver was observed.

In general systemic effects, at 600 mg/kg, decreased body weight gain (up to 58% of control) during the treatment period in both sexes was observed with decreased food consumption (87% of control) in females. Minimal to slight hepatocyte hypertrophy with increased liver weight (up to 1.51-fold over control) in both sex were observed. These findings induced minimal to slight thyroid follicular epithelial hypertrophy and decreased T4 (73% of control) in males. In addition, minimal to slight tubular basophilia in kidneys with increased kidneys weight (up to 1.29-fold over control) and blood urea nitrogen (1.17-fold over control) in males were observed. These findings were considered test item-related adverse effects. Salivation in both sexes at ≥150 mg/kg and increased liver weights in both sexes at ≥35 mg/kg were considered test item-related but not toxicologically relevant.

In clinical pathology, at 600 mg/kg, decreased total red blood cell count (RBC, 92% of control), hemoglobin (HGB, 92% of control), hematocrit (HCT, 91% of control), fibrinogen (FIB, 95% of control) and triglyceride (TG, 41% of control) in males, increased albumin/globulin ratio (A/G, 1.07-fold over control) and gamma glutamyl transpeptidase (GGT, 1.68-fold over control) in males, and increased GGT (2.21-fold over control) in females were observed. At ≥150 mg/kg, prothrombin time (PT, up to 1.13-fold over control) in males was prolonged. These findings considered test item-related but not toxicologically relevant.

 

In conclusion, general systemic adverse effects includinig decreased body weight gain in both sexes, decreased food consumption in females, hepatocyte hypertrophy with increased liver weight in both sexes, thyroid follicular epithelial hypertrophy with decreased thyroxine (T4) in males, and tubular basophilia in kidneys with increased kidney weight and blood urea nitrogen in males were observed at 600 mg/kg.

Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general systemic effects is considered to be 150 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

July 29, 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,6-Octadienal, 3,7-dimethyl-, acid-isomerized

EC Number:

291-768-8

EC Name:

2,6-Octadienal, 3,7-dimethyl-, acid-isomerized

Cas Number:

90480-35-6

IUPAC Name:

1-methyl-4-(propan-2-yl)benzene; ethanol

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley/Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number at receipt: 136 (Male 66, Female 70)
- Number on study: 120 (Male 60, Female 60)
- Age range at receipt: Approximately 7 weeks (Males) and 9 weeks (Females)
- Age range on study: Approximately 10 weeks (Males) and 12 weeks (Females)
- Body weight range: 338.7-404.8 g at the initiation of dosing (Males); 238.0-287.7 g at the initiation of dosing (Females)
- Method of identification: Color marking, Tail tattoo, Cage card
- Health status:Healthy animals were used in this study
- Acclimation: All animals were permitted an acclimation period of 6 days to the laboratory environment
- Animal assignment (Randomization): Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and group assignment), estrus cycle and free from clinical signs of disease or injuries during the acclimation and pre-treatment periods. They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.

HOUSING AND ANIMAL CARE:
- Housing: stainless-steel cage (255W×465L×200H mm)
- Number of animals per cage: Acclimation 2 , Pre-treatment 2 , Treatment 2 or 1, Mating 1 (male):1 (female), Post-mating 2, Recovery 2 or 1, Gestation 1 mated female, Lactation 1 dam with her pups.
- Type of Cage: stainless-steel cage (255W×465L×200H mm) or poly sulfone cages (260W×420L×180 mm) during gestation and lactation.
- Temperature 21.1 - 24.1°C
- Humidity: 40.9 – 57.6%
- Light cycle: 12 hour light/12 hour dark cycle
- Light intensity: 150 - 300 Lux
- Air changes: 10 - 20 times/hour
- Cleaning: Animal room was cleaned daily. Stainless-steel cages were cleaned at least once per 2 weeks, and poly sulfone cages were cleaned at least once per week.
Food, Water and Bedding:
- A standard rat pellet diet was provided to the animals ad libitum. Microbial monitoring for diet was performed
- The animals have had ad libitum access to filtered, ultraviolet light-irradiated municipal tap water at all times. The drinking water was analyzed every 6 months for specified contaminants
- Aspen animal bedding material was sterilized and then provided to the animals in each cage. A certificate of analysis for the bedding was provided by the supplier.
There were no known contaminants in the food, water and bedding material at levels that would be expected to interfere with the results of the study and the data was maintained in the raw data.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose was based on the most recently measured body weight. Animals were dosed at a volume of 2 mL/kg.
Dose formulation was continuously stirred by magnetic stirrer in the animal room (before and throughout the dosing procedure).

The control item or dose formulations were administered by oral gavage once a day at approximately the same time each day (08:30 - 12:30)

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:

One male to one female mating was used, and males and females were paired for 2 weeks. Animals of the recovery group were not mated. Mating confirmation was checked every morning during the mating period. Mating was confirmed with vaginal plug(s) and/or sperm in the vaginal smear.
Day 0 of pregnancy was defined as the day of mating confirmation. Pregnancy was confirmed by implantation sites on the uterus at sacrifice or parturition.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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.

Verification of dose level concentrations:
Analyses of the dosing formulations were conducted and samples for homogeneity analysis were also analyzed for verification of dose level concentration. Results of dose formulations were 107.21, 106.87 and 106.88% at each dose levels of 35, 150 and 600 mg/kg. They were acceptable as the mean concentration was within ±15% of the nominal concentration

Duration of treatment / exposure:

Dosing of the males began 14 days prior to mating and continued throughout mating prior to sacrifice (at least 50 days).
Dosing of the females was beginning 14 days prior to mating and continued throughout lactation day (LD) 13.
Animals of the recovery group were not mated and assigned to 2 weeks of recovery period after the completion of administration.

Frequency of treatment:

Frequency of treatment: 7 days/week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

vehicle control (VC)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

T1

Doses / concentrations 3

Dose / conc.:

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

Remarks:

T2

Doses / concentrations 4

Dose / conc.:

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

Remarks:

T3

No. of animals per sex per dose:

12 animals per sex per group
Additional recovery groups: 0 and 600 mg/kg (6 animals per sex per group)

Control animals:

yes, concurrent vehicle

Details on study design:

120 animals (60 males and 60 females) were included in the study (at the start of administration).
All animals were examined for general health upon receipt. Animals were selected for use in the study on the basis of adequate body weight (recorded on the day of receipt and group assignment). They were randomized and assigned to treatment groups to have a similar mean body weight distribution using the Pristima system based on the most recent body weight.
All animals were permitted an acclimation period of 6 days to the laboratory environment. All assigned animals were permitted a pre-treatment period of 14 days before dosing start.
During the pre-treatment period, a vaginal smear was taken daily for each female and regularity and length of the estrus cycle was examined. Females that failed to exhibit normal estrus cycle were excluded from the study.
Cage card and animal room use record were attached on the cage and animal room, respectively.

- Dose selection rationale:
The dose level was selected based on the results of a two weeks dose range-finding study

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

IN-LIFE OBSERVATIONS AND MEASUREMENTS

Mortality observation:
Mortality and morbidity observations were conducted twice daily except for the acclimation period. In addition, it was conducted once on necropsy day.

General clinical signs:
Clinical signs including mortality, moribundity, general appearance, behavior changes were recorded with date/time of finding, and duration. Animals judged to be abnormal were examined by a veterinarian or an experienced technician.
During the gestation period, dams were especially monitored for signs of abortion or premature delivery. In addition, nursing dams were carefully observed during the lactation period.

Detailed clinical signs:
Detailed clinical observations were made in all animals individually for abnormalities.
Signs noted were included (but are not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition. General clinical signs after dosing were not additionally recorded in the day of detailed clinical signs observation.

Body weight (Period/Frequency):
- Acclimation: animal receipt day and day 6 of acclimation
- Pre-treatment: one time per week
- Treatment: first day of dosing and thereafter one time per week
- Mating: twice a day (before and after dosing)
- Post-mating: twice a day (before and after dosing)
- Gestation and Lactation: twice a day (before and after dosing). However, parturition completed females were observed additionally once more
- Recovery: one time per week
- Necropsy: the day prior to necropsy and on the day of necropsy

Food consumption (Period/Frequency):
- Treatment: one time per week
- Post-mating: one time per week
- Gestation: gestation day (GD) 0, 7, 14 and 20
- Lactation: lactation day (LD) 0, 4 and 13

- Recovery: one time per week
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.

Functional observations:
Sensory function tests (approach and touch response, tail pinch, acoustic startle response and pupillary reflex), grip strength and motor activity were conducted in first six animals per sex in main group (if possible) and all animals in recovery group shortly before scheduled sacrifice.

CLINICAL PATHOLOGY

Blood collection:
Blood samples for clinical pathology were taken from all scheduled sacrifice animals in main and recovery groups. Blood samples were collected from abdominal vena cava.

Hematology:
Approximately 1.5 mL of blood samples was collected from all scheduled sacrifice animals in main and recovery groups.
Hematology Parameters:
Total leukocyte count (WBC), Mean corpuscular hemoglobin (MCH), Total red blood cell count (RBC), Mean corpuscular hemoglobin concentration (MCHC), Hemoglobin (HGB), Platelet count (PLT), Hematocrit (HCT), Reticulocyte count, Mean corpuscular volume (MCV), WBC differential count, Prothrombin time (PT), Activated partial thromboplastin time (APTT), Fibrinogen (FIB).

Clinical chemistry:
Approximately 1.5 mL of blood samples was collected from all scheduled sacrifice animals in main and recovery groups.
Clinical Chemistry Parameters:
Glucose (GLU), Alanine aminotransferase (ALT), Blood urea nitrogen (BUN), Total bilirubin (TBIL), Creatinine (CREA), Alkaline phosphatase (ALP), Total protein (TP), Gamma glutamyl transpeptidase (GGT), Albumin (ALB), Creatine phosphokinase (CK), Albumin/globulin ratio (A/G), Calcium (Ca),
Total cholesterol (TCHO), Inorganic phosphorus (IP), Triglyceride (TG), Sodium (Na), Phospholipid (PL), Potassium (K), Aspartate aminotransferase (AST), Chloride (Cl).

Thyroid Hormone (T4 and TSH) Analysis:
Blood samples (approximately 0.6 mL) for thyroid hormone (T4 and TSH) analysis were collected.
Pups blood was pooled in a tube by sex per litter. Thyroid hormone (T4 and TSH) concentration in the serum samples of pups on PND 13 were analyzed.

Parturition monitoring:
Mating-proven females were monitored twice daily for signs of parturition including abortion, premature delivery and difficult or prolonged parturition from GD 21.
Dams were allowed to litter. The duration of gestation, the number of dead and live pups, runts, sexing of live pups, individual body weight of live pups and external abnormalities were observed.
Females which were not observed to have parturition until GD 27 were sacrificed.

Oestrous cyclicity (parental animals):

A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating.
Furthermore, regularity and length of the estrus cycle was examined during the treatment period until mating.
In addition, vaginal smear of sacrificed females was taken at termination to examine the stage of the estrus cycle and allow correlation with histopathology of female reproductive organs.

Sperm parameters (parental animals):

not specified

Litter observations:

Pups mortality:
All pups were monitored for mortality and morbidity once every day.

Pups general clinical signs:
All pups were monitored for general clinical signs once every day. Clinical signs including general appearance and behavior changes were recorded.

Pups body weight:
Body weight and sex were recorded on post-natal day (PND) 0, 4 (before culling), and 13, and it was reported as litter.

Anogenital distance and nipple retention:
Anogenital distance was measured in all pups on PND 4. Both, 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. In addition, the number of nipples in all male pups was counted on PND 12.

Pups culling:
On LD 4, the size of each litter was adjusted by eliminating extra pups randomly to give 4 males and 4 females per litter, if possible. Litters with 8 or fewer pups were not culled. External examinations were conducted on all culled pups and subsequently humanly sacrificed by CO2 inhalation. Special attention was paid to the external reproductive genitals which should be examined for signs of altered development.

Postmortem examinations (parental animals):

Unscheduled Sacrifice:

Macroscopic findings:
Complete necropsy was performed under the direct supervision of a veterinarian pathologist. After blood sampling, the animals were sacrificed by exsanguination from the vena cava and aorta. The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial
cavities were examined for abnormalities and the organs were removed and examined.

Tissue preservation:
Tissues from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative and the testes and epididymides which were fixed in Bouin’s fixative. The tissues were placed in the appropriate fixative for approximate 48 hours, an d then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder. Tissues from unscheduled sacrificed animals were processed for microscopic evaluation.

Scheduled Sacrifice:
Method of termination
All surviving main group males were euthanized on the day after final dosing using isoflurane.
All surviving main group females on LD 14 were euthanized using isoflurane.
All surviving recovery group males and females were euthanized using isoflurane at least 14 days after the first scheduled sacrifice of males and females.
All animals were fasted more than 16 hours (overnight) prior to scheduled sacrifice.

Macroscopic findings:
Complete necropsy was performed under the direct supervision of a veterinary pathologist. After blood sampling, the animals were sacrificed by exsanguination from the vena cava and aorta.
The animals were examined carefully for external abnormalities. The abdominal, thoracic and cranial cavities were examined for abnormalities and the organs were removed and examined.

Organ weights:
Organs were weighed for all animals at terminal and recovery sacrifice groups, and organ/body weight ratios using the terminal body weight (TBW) obtained prior to necropsy were calculated. However, paired reproductive organs were weighed separately.

Tissue preservation:
Tissues from each animal were preserved in 10% neutral buffered formalin, except the eyes (with optic nerve) which were fixed in Davidson’s fixative and the testes and epididymides which were fixed in Bouin’s fixative.
The tissues were placed in the appropriate fixative for approximate 48 hours, and then transferred to 70% ethanol. Formalin was infused into the lung via the trachea and into the urinary bladder.

Microscopic findings:
Tissues except reproductive organs and thyroids (with parathyroids) collected from the first six animals per sex in the main group were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. In addition, all reproductive organs (ovaries, prostate, testes,
epididymides, uterus with cervix and seminal vesicles with coagulation gland) and abnormal lesions collected from all animals in the main group were further processed to slides, stained with H&E, and examined microscopically. Thyroids (with parathyroids) for adult males were further processed to slides, stained with hematoxylin and eosin, and examined microscopically to clarify the change of thyroid hormone. Any target organs were additionally evaluated microscopically from recovery group animals.

Postmortem examinations (offspring):

Dead pups:
External and visceral examinations were conducted in dead pups at parturition and dead or moribund pups from birth to day 4 of lactation, if possible.
Special attention was paid to the external reproductive genitals which should be examined for signs of altered development.

Statistics:

Mean values and standard deviations were calculated in the final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima
System or Statistical Analysis Systems.
Data from non-pregnant females until mating will be included in the report. Data were 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.
For comparing control group and recovery group, the data was analyzed for homogeneity for variance using F-test.
Homogeneous data was analyzed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test.
Heterogeneous data was analyzed using Kruskal-Wallis Test and significant difference between control and recovery group was assessed using Dunn’s Rank Sum Test.
Data presented as frequencies was analyzed by χ2-test followed by the Fisher's exact test where necessary.

Reproductive indices:

Based on the results, the following reproduction indices were calculated:

- Mating Index (%) = (No. of males with evidence of mating / No. of males paired) × 100
= (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
= (No. of pregnant females / No. of females paired) × 100

- Fecundity Index and Pregnancy Index (%) = (No. of males impregnating a female / No. of males with evidence of mating) × 100
= (No. of pregnant females / No. of females with evidence of mating) × 100

- Precoital Time: No. of days taken to mate

Offspring viability indices:

Based on the results, the followings were calculated:

On Day 0 of Lactation
- Delivery Index = (No. of dams with live pups / No. of pregnant dams) × 100

On Day 4 of Lactation
- Viability Index = (No. of live pups on Day 4 of lactation / No. of live pups at birth) × 100

On Day 13 of Lactation
- Weaning Index = (No. of live pups on Day 13 of lactation / No. of pups on Day 4 of lactation after culling) × 100

Dead pups:
External and visceral examinations were conducted in dead pups at parturition and dead or moribund pups from birth to day 4 of lactation, if possible.
Special attention was paid to the external reproductive genitals which should be examined for signs of altered development.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In both sexes at ≥ 150 mg/kg, salivation was observed.
Other clinical signs during the study were not considered test item-related since these findings were observed with low frequency, occurred sporadically, generally of low severity and/or did not have a dose-response.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One female (Animal No. 115) in the recovery group at 600 mg/kg was found dead on dosing day 47.
Macroscopic findings included minimal enlarged adrenal glands and partial irregular surface of the right kiney. Microscopic analysis revealed marked pyelonephritis in the kidneys, severe necrosis in the adrenal glands and slight hepatocyte hypertrophy in the liver.
Unscheduled sacrifice was performed on eight females (Animal No. 103, 105, 106, 108, 110, 112, 113 and 114) at 600 mg/kg .
In the results of microscopic findings, minimal to slight hepatocyte hypertrophy in the liver was observed in some unscheduled sacrificed animals, but there were no changes in reproductive organs.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at 600 mg/kg, decreased body weight gain in the main group (89% of control) and recovery group (79% of control) during the treatment period was observed.
In females at 600 mg/kg, decreased body weight gain in the main group (85% of control) and recovery group (58% of control) during the treatment period was observed.
Other statistically significant change in body weight was not considered test-item related since it was transient.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In females at 600 mg/kg, decreased food consumption in the main group (89% of control) during the treatment period was observed.
Other statistically significant changes in food consumption were not considered test-item related since it was minimal or transient.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
In males at 600 mg/kg, decreased total red blood cell count (RBC, 92% of control), hemoglobin (HGB, 92% of control), and hematocrit (HCT, 91% of control) were observed.
Other statistically significant changes in hematology were not considered test item-related since it was minimal or only observed in the recovery group.

Coagulation:
In males at ≥ 150 mg/kg, increased prothrombin time (PT, up to 1.13-fold over control) and at 600 mg/kg decreased fibrinogen (FIB, 95% of control) were observed.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males at 600 mg/kg, increased blood urea nitrogen (BUN, 1.17-fold over control), albumin/globulin ratio (A/G, 1.07-fold over control) and gamma glutamyl transpeptidase (GGT, 1.68-fold over control) and decreased triglyceride (TG, 41% of control) were observed.
In females at 600 mg/kg, increased GGT (2.21-fold over control) was observed.
Other statistically significant changes in clinical chemistry were not considered test item-related because there no dose-relationship, no correlated microscopic changes and/or observed only in recovery group.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related changes in functional behavior examination were observed in both sexes during the study.

Motor activity examination:
No test item-related changes in motor activity examination were observed in both sexes during the study.
A statistically significant change in motor activity examination was not considered test item-related since it was transient and did not have a dose-response.

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):

In both sexes at 600 mg/kg, minimal to slight hepatocyte hypertrophy in liver was observed.
In males at 600 mg/kg, minimal to slight follicular epithelial hypertrophy in thyroid glands was observed.
In addition, minimal to slight tubular basophilia in kidney and minimal to slight ductal atrophy in epididymides were observed.
Other changes observed in microscopic findings were considered incidental or spontaneous changes since they were infrequent, generally of low severity, and similarly distributed among control and treated groups.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid Hormone (T4 and TSH) Analysis: In males at 600 mg/kg, decreased T4 (73% of control) was observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

At 600 mg/kg, two females with irregular estrus cycle were observed.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- Precoital time:
No test item-related changes in precoital time were observed during the study.

- Fertility data:
At 600 mg/kg, decreased fertility index (33% of control), fecundity index (40% of control) and pregnancy index (40% of control) were observed.

Details on results (P0)

At 600 mg/kg, decreased number of implantation (63% of control), pups born (65% of control), live litter size from PND 0 to 13 (up to 58% of control), viability index (94% of control) and weaning index (97% of control) were observed. Decreased pups born and live litter size from PND 0 to 13 were considered secondary effect since the number of implantations was decreased.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

reproductive function (oestrous cycle)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related changes in F1 pups clinical signs were observed in both sexes during the study.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 600 mg/kg, decreased F1 male and female pup body weight (up to 59% of control) were 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:

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):

F1 culled pups external examination:
No test item-related changes in F1 culled pups external examination were observed in both sexes during the study.

F1 pups external examinations:
No test item-related changes in F1 pups external examinations were observed in both sexes during the study

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- F1 pups anogenital distance:
No test item-related changes in F1 pups anogenital distance were observed in both sexes during the study.

- F1 male pups nipple retention:
No test item-related changes in F1 male pups nipple retention were observed during the study.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general reproductive/developmental effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

600 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The No-Observed-Adverse-Effect Level (NOAEL) for reproductive/developmental effects when the test item is administered via oral gavage to Sprague-Dawley rats with dose levels of 0, 35, 150 and 600 mg/kg bw/day is considered to be 150 mg/kg/day for the animals of both sexes.

Executive summary:

This study was conducted to evaluate the potential reproductive/developmental toxicities of the test item when administered via oral gavage to Sprague-Dawley rats. The assay was performed in accordance to OECD TG 422 and in compliance to GLP.

The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 35, 150 and 600 mg/kg with a dose volume of 2 mL/kg. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (total 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in the recovery groups 0 and 600 mg/kg (6 animals per sex per group) received the test item but were not mated. Afterwards, they were assigned to 2 weeks of recovery period after the completion of the test item administration.

Reproductive/developmental observations including estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were measured. Thyroid hormone (T4 and TSH) level in blood was also analyzed for pups at sacrifice.

Eight females at 600 mg/kg were sacrificed unscheduled due to non-parturiton and non-mated. This resulted in a decreased fertility, fecundity and pregnancy index. In addition, reproductive and littering finding changes including decreased number of implantation, pups born, live litter size from PND 0 to 13, viability index and weaning index. These findings were considered attributed to microscopic findings of mated males which revealed ductal atrophy in epdididymides since there were no test item-related changes in female reproductive organs. These findings were considered as test item-related adverse effects. Body weight gain was decreased during the treatment period in both sexes at 600 mg/kg. Decreased food consumption during the treatment period in females at 600 mg/kg was also found. These changes were considered to be accompanied by decreased F1 pups body weight. These findings were considered test item-related adverseeffects.

Two females with irregular estrus cycle were observed at 600 mg/kg. The relationship of test item and this finding was uncertain since the incidence was ambiguous. However, it was not considered to have toxicological relevance since no test item-related changes in microscopic findings of female reproductive organs as well as other females at 600 mg/kg.

Reproductive/development adverse effects including ductal atrophy in epdididymides, decreased fertility, fecundity and pregnancy index in both sexes, reproductive and littering finding changes including decreased number of implantation, pups born, live litter size from PND 0 to 13, viability index, weaning index, and decreased F1 pups body weight were observed at 600 mg/kg.

Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for reproductive/developmental effects is considered to be 150 mg/kg/day.