Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 Oct 2010 to 25 Mar 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This species and strain of rat are commonly used for general toxicity studies and background data for them was available at the study facility as well.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Hino Rearing Center
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 316.6 to 358.1 g for males and 204.0 to 231.1 g for females. The weights at day 1 of administration were confirmed to fall within the mean weight ± 20% range for males and females in each group.
- Fasting period before study: No
- Housing: Stainless steel wire-bottom cages were used
- Diet (e.g. ad libitum): ad libitum (MF, Lot number 10034, Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): ad libitum (chlorinated Hita municipal tap water)
- Acclimation period: yes, 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 to 24.4°C
- Humidity (%): 49.2 to 65.7%
- Air changes (per hr): ventilation frequency of 10 to 15 times/hour
- Photoperiod (hrs dark / hrs light): light/dark cycle of 12-hour intervals (lights on at 7:00 and off at 19:00)

IN-LIFE DATES: From: October 19 to November 30, 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
he test substance was weighted accurately and a test solution (suspension) of 7.50 w/v% was prepared by adding a constant volume of olive oil to it. Then, part of the 7.50 w/v% test substance was collected while stirring with a magnetic stirrer and olive oil was added to dilute the solution to prepare test solutions (suspension) of 2.50 and 0.750 w/v%. Test solution at each concentration was divided into plastic containers and then stored in a cool place (3-9°C, allowable range 1-10°C). On each administration day, test solution of each concentration was taken from the storage location for use. Test solutions were used within 15 days of preparation.

VEHICLE: olive oil
- Rationale: Based on trial formulations performed at facilities.
Details on mating procedure:
After 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group. Females were returned to their original cages in the morning of the following day. Detection of mating was confirmed by evidence of sperm in the vaginal smear or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The "Homogeneity, stability, and concentration confirmation study on a test solution of 6,6'-di-tert-butyl-4,4'-diethyl-2,2'-methylenediphenol" (study code: X02-0238) conducted at this test facility using high speed liquid chromatography (HPLC) confirmed the homogeneity of the test solution of 0.0100 and 25.0 w/v% and the stability of the test solution when stored in a cool place.
Immediately after preparing the test solution, n=1 samples were taken from each of the top, middle and bottom layers. After storage in a cool place for 3, 7, and 16 days, n=1 samples were taken from the middle layer and after pre-processing, HPLC was used to measure test substance concentration once. The results showed that the test solution was homogeneous and that the concentration of the test substance was 0.0106 w/v% for the 0.0100 w/v% solution and 25.0 w/v% for the 25.0 w/v% solution 16 days after preparation, which were both within the criteria of the study facility, the test solution was deemed stable for 15 days stored in a cool place.
Duration of treatment / exposure:
Males were exposed for 42 days for the main study group and recovery group, as well as the female recovery group. Females were exposed for a period of 42 to 49 days, i.e. through mating, pregnancy, parturition, and the 4-day postpartum period including the nursing period. The mating period was from the 15th day of administration for up to 14 days. For the pregnancy period, the day that copulation was confirmed was treated as day 0 of pregnancy and the pregnancy period was considered to be over on the day prior to the completion of parturition. For the nursing period, the day of parturition was treated as day 0 of nursing and the nursing period continued for 4 days thereafter. Males and females both had a 14-day recovery period after the 42-day administration period.
Frequency of treatment:
Once daily between 9:00 and 13:55
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels were based on the results of the dose setting study (study code C21-0034, non-GLP study) for "Combined reproductive/developmental toxicity study and study of toxicity of repeat oral administration of 6,6’-di-tert-butyl-4,4’-diethyl-2,2’-methylenediphenol in rats.” The test substance was orally administered repeatedly over a 14-day period to 3 male and 3 female 9-week-old Crl:CD (SD) rats per group at doses of 0 (olive oil), 25, 250, 500, and 1,000 mg/kg/day. General status observations, weight measurement, hematologic tests, blood biochemistry tests, organ weight measurement, and autopsy were conducted.
Study design: The administered dose was 0 (olive oil), 30, 100, and 300 mg/kg/day and 12 each of males and females were placed in each group. Also, 5 of the 12 males in the control and high dose group and 5 additional females that were not allowed to mate were set to be part of the recovery group. Further details about the study design are given in Table 1 below.
Method: Oral gavage, using a syringe (Terumo) outfitted with a soft catheter (Terumo)
Dose volume: 4.0 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
Mortality / Viability At least twice daily before and after administration

CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were made twice daily before and after administration, and whenever an animal's status grew worse for all animals during the administration period. During the recovery period, observations of general status were made once daily. Also, mothers were observed for parturition status and nursing status. Observations were also taken on the day of autopsy. Detailed observations (animal's response when removed from cage, detailed observations when held in hands and observations of movement in the arena) were made for all animals prior to the start of administration, and once a week during the administration period. During the mating period, males and females were not observed.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: 1st, 3rd, 7th, 14th, 21st, 28th, 35th, and 42nd day of administration. Females: For the recovery group the same days as for males. For the main study group, the same days as the males up to mating confirmation and then on days 0, 7, 14, 17, and 20 of pregnancy after mating confirmation and on days 0 and 4 of nursing after parturition

FOOD CONSUMPTION: Yes
Males/Females: 1st, 3rd, 7th, 14th, 28th, 35th, and 42nd day of administration. Food consumption of mated females was measured on Days 0, 7, 14, 17, and 20 of pregnancy after mating confirmation and on days 0 and 4 of nursing after parturition. During the recovery period the foodconsumption was measured 1, 3, 7 and 14 days after the final dose for both males and females.

WATER CONSUMPTION: No

FUNCTIONAL OBSERVATIONS: Yes
The following tests were performed on the selected 5 animals/sex/group (For males in the 300 mg/kg group in which deaths were observed, 4 animals were selected):
- Vision and hearing ability, pain, pupillary reflex, static righting reflex.
- fore- and hind-limb grip strength, recorded as the mean of two measurements per animal (Grip strength meter FGC-2 Matis).
- Amount of sponteneous movements: A movement measuring device for rats, ACTIMO-10, (Shintechno Corporation) was used to measure the amount of movement of the animals over a 1-hour period (6 times in 10-minute intervals) and the number of times the infrared lines (in a crisscross pattern every 5 cm in a 42.6 cm x 26.5 cm area) were crossed was used for evaluation.

The selected males and females of the recovery group were tested during Week 6 of treatment. The females of the recovery group were also tested during Week 6 of treatment. The female main study group was tested once prior to fasting on the 4th day of nursing by selecting 5 animals with the closest dates of parturition from among parturient animals. Responsiveness and grip strength were tested in the same manner as that used in detailed observations of general status to conceal the study group. Tests were not conducted during the recovery period because tests conducted during the administration period showed no changes that suggested an effect of test substance administration.

BLOOD: Blood samples were collected at the end of the treatment period/recovery period on the day of scheduled necropsy from the selected 5 animals/sex/group (4 males in the 300 mg/kg group) under anaesthesia. The animals were fasted (for 16-20 hours) before blood sampling.
Blood samples were drawn from the abdominal aorta and collected into tubes prepared with K2-EDTA for haematological parameters (whole blood), with citric acid trisodium dihydrate for clotting tests and tubes treated with sodium heparin for biochemistry parameters (plasma). Serum was obtained by collecting blood into a glass test tube and then subjecting it to centrifugal separation

HAEMATOLOGY: The following haematology parameters were determined in blood:
Red blood cell count (RBC), White blood cells (WBC), Hemoglobin concentration (Hb), Hematocrit value (Ht), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin volume (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Reticulocyte count/percentage (Reticulo), Leukocyte differential (Differentiation of leukocyte), Neutrophils (Neutro), Eosinophils (Eosino), Basophils (Baso), Lymphocytes (Lymph), Monocytes (Mono), Large unstained cells (LUC),

CHEMICAL BIOCHEMISTRY: Yes
Serum was used to measure the following parameters: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), γ -glutamyl transpeptidase (γ-GTP), Total cholesterol (T-Cho), Triglycerides (TG), Blood glucose (Glucose), Total protein (T-Protein), Albumin (Albumin), Phospholipids (PL), Lactic acid dehydrogenase (LDH), A/G ratio (A/G ratio), Blood urea nitrogen (BUN), Creatinine (Creatinine), Total bilirubin (T-Bil), Total bile acids (TBA), Calcium (Ca), Inorganic phosphorus (IP), Sodium (Na), Potassium (K), Chloride (Cl)
Oestrous cyclicity (parental animals):
Daily vaginal smears were taken from all female, including those in the recovery group to determine the stage of estrous from the day following the day of acquisition until the 14th day of treatment. Giemsa staining was performed and miscopy tests were done. The duration of the estrous cycle was counted from estrous to the following estrous and the mean estrous cycle was calculated. If estrous continued for 2 or more days, the first estrus was treated as the day of estrous. Also, the rate of individuals showing abnormal estrous cycles (individuals not showing regular estrous cycles) was calculated.
Litter observations:
Each litter was examined to determine the following, if practically possible:
- Mortality / Viability: After parturition (nursing day 0), the number of births (total number of stillbirths and live births), the number of live births (total number of surviving and dead pups), sex, and appearance were observed. Also, the dead pup and stillbirth distinction was made by investigating the lungs. If traces of pulmonary respiration were present, it was considered a dead pup. If absent, it was considered a stillborn animal. Also, the body surface, orifices, thoracic cavity, and abdominal cavity of dead pups and stillborn animals were macroscopically observed.
- Clinical signs: Observations of general status, including death, and determination of sex were conducted once daily up to nursing day 4.
- Body weights: On nursing day 0 and 4, the weights of surviving pups were measured for male and female animals for each litter.
- Sex: Sex was determined for all pups on nursing day 0 and once daily up to nursing day 4



Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of implantation sites were recorded for all paired females.
Organ and tissue samples shown in the following table were collected from all animals:
Respiratory system Trachea, lungs
Digestive system Stomach, intestines (including the section from the duodenum to the rectum and Peyer patch), liver*
Cardiac/vascular system Heart*
Urinary system Kidneys*, bladder
Reproductive system Testicles*, epididymis*, prostate, seminal vesicles, ovaries*, uterus*, vagina
Nervous system Brain* (including the cerebrum, cerebellum, and pons), spinal cord, sciatic nerve
Hematopoietic system Bone marrow (femur), axillary lymph nodes, mesenteric lymph nodes, spleen*, thymus*
Endocrine system Pituitary*, thyroid* (including parathyroid gland), adrenal gland*
Sensory system Eyes

Organs and tissues indicated with an *were weighed using an electronic balance (Sartorius AG).

HISTOPATHOLOGY: Yes
The preserved organs and tissues of selected 5 animals/sex as indicated in Table 2 below were examined. All abnormalities were described and included in the report.
Postmortem examinations (offspring):
MACROSCOPIC EXAMINATION
On day 4, the body surface, orifices, intracranial cavity, thoracic cavity, abdominal cavity, and pelvic cavity of surviving animals were macroscopically observed.
Statistics:
The following statistical methods were used to analyze the data:
- The results were tested for homogeneity of variances in each group using the Bartlett test. Items for which there were no significant differences at the 5% level with the Bartlett test and items that were deemed to have equal variances were tested using one-way analysis of variance. If differences were observed at the 5% level, the Dunnett test was conducted between the control group and test substance groups for each dose. Parameters for which significant differences were found at the 5% level with the Bartlett test and deemed to have unequal variances were tested using the Kruskal-Wallis method and if significant differences were found at the 5% level, further tests were conducted between the control group and test substance groups for each dose using the non-parametric Dunnett test.
The incidence of abnormal estrous cycle, the rate of copulation, the rate of fertilization, the rate of conception, and the birth rate for parent animals, as well as the ratio of the sex, the day 0 surviving animal sex ratio and the day 4 surviving animal sex ratios of newborn animals were tested for significant differences as compared to the control group using the Fisher's exact test.
Offspring viability indices:
Obtained test results were used to calculate the means and standard deviations for the number of male/female births and the total number of births, including dead pups on day 0 and stillbirths, the number of stillbirths, the number of male/female live births, including deaths on day 0 of nursing, the total number of live births, the number of surviving male and female animals on nursing day 0 and 4 and totals thereof, as well as the weight by sex. Also, general status and autopsy results were recorded. The number of animals of each sex was found for births and live births Also, the means and standard deviations for the following items by litter and by dose were calculated. The rate of external abnormalities by type was not calculated.
-Birth rate: (number of live births/number of implantation sites)x100
-Born animal sex ratio: Number of male animals born/number of births
-Day 0 survival rate: (number of live births/number of births)x100
-Day 0 surviving animal sex ratio: Number of surviving male animals on day 0/number of surviving animals on day 0
-Rate of external abnormalities: (number of live births with external abnormalities/number of live births)x100
-Day 4 survival rate (number of surviving animals on day 4/number of live births)x100
-Day 4 surviving animal sex ratio: Number of surviving male animals on day 4/number of surviving animals on day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males and females in the 300 mg/kg group had loose stool, diarrhea, hypersalivation, spontaneous movement reduction, reduced respiratory frequency, and partial eyelid opening and females had mucous stool, reduced fecal volume, reduced food intake, and rough fur. Males and females in the 100 mg/kg group had diarrhea and hypersalivation. For males in the 300 mg/kg group, detailed observations of general status found reduced body temperature, fur soiling, pallor, lacrimation, and mild respiratory insufficiency in 1 male in the 3rd week of administration. For females, fur soiling was observed in the 3rd week of administration, lacrimation and hypersalivation were observed in the 4th week of administration, and hypersalivation was observed on day 4 of nursing in 1 animal each.
Mortality:
mortality observed, treatment-related
Description (incidence):
Three of the males in the 300 mg/kg group were moribund and 4 died. Also, 5 of the females in the same group and 2 of the females in the 100 mg/kg group died during late pregnancy or immediately after giving birth.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated animals decreased for both males and females of the highest dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced food intake was observed in females of the highest dose group.
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):
Prolonged prothrombin time and activated partial thromboplastin time were observed in the males in the 100 mg/kg and higher groups, a trend towards prolonged activated partial thromboplastin time was observed in females in the 300 mg/kg group, reduced red blood cell count, hemoglobin concentration, and hematocrit value and elevated platelet count were observed in males in the 300 mg/kg group, and reduced hemoglobin concentration and hematocrit value were observed in the females in the 300 mg/kg recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood biochemistry tests showed elevated alanine aminotransferase values in females in the 300 mg/kg group and elevated γ-glutamyl transpeptidase values in females in groups of 100 mg/kg and higher.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
1) During the administration period higher values in sponteneous movement were observed in males at 50 to 60 minutes in the 100 mg/kg group, but these changes were not dose dependent. No abnormalities were observed in the 30 and 300 mg/kg groups.In females, no abnormalities were observed in any test substance groups.
2) During the recovery period neither males nor females had any changes suspected to have been affected by the test substance during the administration period, so tests were not conducted during the recovery period.
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):
Males in groups of 100 mg/kg and higher and females in groups of 300 mg/kg and higher had centrilobular hepatocyte hypertrophy, diffuse thyroid follicular epithelial cell hypertrophy and that females in the 300 mg/kg group had ground-glass changes in hepatocytes.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Although not statistically significant, 1 animal in the 300 mg/kg group had a continuous resting phase. No abnormalities were observed in the 30 and 100 mg/kg groups.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
spermatogenic staging profiles were normal for all males examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All animals in all administration groups, including the control group had copulation established and infertile animals were not found. Also, while no abnormalities were observed in the days required for copulation, in the 300 mg/kg group, the corpus luteum graviditatis count and implantation site count were low and the parturition rate tended to be low. Additionally, in the 100 mg/kg group, low implantation rate and high pre-implantation loss rate was observed and in the 300 mg/kg group, a trend towards high post-implantation loss rate was observed, but these changes were not dose dependent. No abnormalities were observed in the 30 mg/kg group.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental systemic
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
haematology
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
In the 300 mg/kg group, reduced number of births and a trend towards reduced number of live births, day 0 surviving pups, and birth rate was observed. The postnatal survival rate on Day 4 in each dose group was 77.3% (control), 82.6% (30 mg/kg), 99.3% (100 mg/kg) and 76.7% (300 mg/kg). Additionally, 1 pup in the control group had tail loss and pups without milk bands and reduced body temperature were observed in all dose groups, including the control group. In the 100 mg/kg group, paleness was observed in 1 animal. However, none of these changes were dose dependent.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males, no abnormalities were observed on day 0 of nursing in the test substance administration groups, but a trend towards reduced weight was observed on day 4 of nursing in the 300 mg/kg group. In females, reduced values were observed on day 4 of nursing in the 300 mg/kg group.
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:
effects observed, non-treatment-related
Description (incidence and severity):
White parts on the liver were observed in 2 animals in the 300 mg/kg group and in 1 animal in the 100 mg/kg group, but because there were few cases, these changes were deemed incidental. Additionally, tail loss was observed in the control group and pyelectasis of the kidneys was observed in the 30 mg/kg group, while the 100 mg/kg group had kidney pyelactasis and ureter extension, but none of these changes were dose dependent.
Histopathological findings:
not examined

Details on results (F1)

The postnatal survival rate on Day 4 in the 300 mg/kg group was 76.7%, but it was reduced in both the control group and 30 mg/kg group, so dose dependence was unclear. However, the day 4 survival rate in the 100 mg/kg group was 99.3% and in addition to all pups dying in the 300 mg/kg group, deaths were observed during the nursing period, so the day 4 survival rate was reduced. For cases of all pups dying in the 30 mg/kg group, dose dependence was not observed, so animal handling was the most likely cause. However, mothers in the 300 mg/kg group had diarrhea, low weight, and other degradations of their condition and newborn animals also had low weight, so the low day 4 survival rate was deemed to have been caused by test substance administration.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects

Applicant's summary and conclusion

Conclusions:
A NOAEL of 30 mg/kg bw for systemic effects and a NOAEL of 100 mg/kg bw for reproductive toxicity was established in a combined oral repeated dose toxicity study with reproduction/developmental toxicity screening test (OECD 422, GLP).
Executive summary:

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of 6,6`-di-tert-butyl-4,4`-diethyl-2,2`-methylenediphenol in rats by oral gavage was performed according to OECD 422 and under GLP conditions. Based on the results of a 14-day dose range finding study, dose levels of 30, 100 and 300 mg/kg were selected for this study. The test item, formulated in olive oil, was administered daily by oral gavage to Sprague-Dawley rats. One control group and three treated groups were tested, each consisting of 12 males and 12 females. For the control and highest dose group, 5 animals of each sex that were not allowed to mate were set to be part of the recovery group.

Males were exposed for 42 days for the main study group and the recovery group, as well as the females from the recovery group. Females were exposed for a period of 42 to 49 days, i.e. 2 weeks through mating, pregnancy, parturition, and the 4 -day postpartum period including the nursing period.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity, body weight and food consumption (at least at weekly intervals), estrous cycle determination, clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).

Formulations were analyzed once during the study to assess accuracy and homogeneity and stability. Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Parental results: Three of the males in the 300 mg/kg group were moribund and 4 died. Also, 5 of the females in the same group and 2 of the females in the 100 mg/kg group died during late pregnancy of immediately after giving birth. Body weights and body weight gain decreased for both males and females of the highest dose group and reduced food intake was observed in females. Blood biochemistry tests showed elevated alanine aminotransferase values in females in the 300 mg/kg group and elevated γ-glutamyl transpeptidase values in females in groups of 100 mg/kg and higher. Prolonged coagulation times were observed in males at 100 mg/kg and higher. Reduced red blood cell count, hemoglobin concentration, and hematocrit value and elevated platelet count were observed in males in the 300 mg/kg group, and reduced hemoglobin concentration and hematocrit value were observed in the females in the 300 mg/kg recovery group.

As for organ weights, males in groups of 30 mg/kg and higher had elevated absolute and relative weights of liver and females in groups of 100 mg/kg and higher had elevated absolute and relative weights of thyroid and liver. Histopathological tests showed that males in groups of 100 mg/kg and higher and females in groups of 300 mg/kg and higher had centrilobular hepatocyte hypertrophy, diffuse thyroid follicular epithelial cell hypertrophy and that females in the 300 mg/kg group had ground-glass changes in hepatocytes.

Reproductive toxicity results: Mothers showed a trend towards reduced corpus luteum graviditatis count and implantation site count, as well as reduced parturition rate in the 300 mg/kg group. For the pups, there was a reduced number of births from animals in the 300 mg/kg group, as well as a trend towards reduced number of live births, number of surviving births on day 0, and birth rate. Also, a trend towards reduced survival rate on day 4 of nursing was observed in the same group, as were low weights and a trend towards reduced weight.

Based on the above results, the following No Observed Adverse Effect Levels (NOAELs) were derived.

- Parental systemic NOAEL: 30 mg/kg

- Reproduction NOAEL: 100 mg/kg