Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28.03.2017-21.07.2017
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:
28.03.2017-21.07.2017
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
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Specific Pathogen Free
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc., 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
- Age at the start of administrationn: 10 wks (males), 12 wks (females)
- Weight at the start of administration: Males: 353.4–406.6 g, Females: 216.7–288.1 g
- Fasting period before study: not specified
- Housing: Stainless wire mesh cages, 260W×350D×210H (mm); Polycarbonate cage, 260W×420D×180H (mm)
- Number of animals per cage: two animals (during quarantine-acclimation period); one animal (during dosing period)
- Diet (e.g. ad libitum): ad libitum; pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water (e.g. ad libitum): ad libitum; public tap water, filtered and irradiated by ultraviolet light
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1–24.4°C
- Humidity (%): 39.0–60.7%
- Air changes (per hr): 10–15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
Route of administration:
oral: gavage
Details on route of administration:
Animals were randomly assigned to groups in an attempt to equalize mean group body weights.
A temporary identification number was marked on the tail using a red indelible pen.

Individual doses were calculated for each animal based on the most recently
recorded body weight data at a dose volume of 2 mL/kg. Dosing solutions were
administered once daily using 1 or 3 mL disposable syringes fitted with a gastric
intubation tube.
Control animals were dosed with vehicle only (corn oil).
Vehicle:
corn oil
Details on oral exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks
prior to mating, during 2 weeks of mating and 21 days of post-mating).
Also, males and females of the recovery groups were dosed once daily for 49 days.
Females of the main group were dosed once daily for 2 weeks prior to mating until
Postpartum Day 13. Also, females showing no evidence of parturition signs were
dosed until Gestation Day 25.
The control group animals were dosed with the vehicle only with the same dose
volume as the test substance-treated groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the treatment period, formulation samples were prepared and analysed for stability and
homogeneity of the test item in the selected vehicle.
Verification of dose level concentrations were conducted using Gas Chromatography
Duration of treatment / exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating).
Also, males and females of the recovery groups were dosed once daily for 49 days.
Females of the main group were dosed once daily for 2 weeks prior to mating until Postpartum Day 13.
Also, females showing no evidence of parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
7 days per week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
- 12 animals per sex per group
- additional 6 animals per sex per group for the recovery groups (control and high dose)
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.
Body weights were recorded on the days of receipt, the last day of the quarantine-acclimation period and the day of group assignment.
All animals were quarantined and acclimated for 7 days and observed once daily for general health condition and clinical signs.
After the quarantine-acclimation period, all animals were observed once daily for clinical signs until group assignment.
Following group assignment, animals were uniquely identified by a blue indelible marking on the tail. A color-coded cage card was placed on each animal’s cage
displaying the group and dose level information.
Animals were randomly assigned to groups in an attempt to equalize mean group body weights.

- Dose selection rationale:
According to the results of a previous dose range finding study the doses were selected for the 3 dose groups.

- Body weights:
-- Body weights of males of the main group and animals of both sexes of the recovery groups 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 prior to 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 (the first day of dosing), once a week throughout the dosing period, the day prior to necropsy and on the day of necropsy.
Fasted body weights recorded on the day of necropsy were presented, but were not included in the statistical analysis.

- Food consumption:
-- Food consumption of males of the main group and animals of both sexes of the recovery group was recorded just prior to dosing on Day 0 (one day before first
dosing), once a week during the dosing and recovery periods (except during mating) and the day prior to necropsy.
-- Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before first dosing), once a week throughout the dosing period and the day prior to necropsy. Residual feed was recorded on the next day.
Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

Positive control:
not applicable
Observations and examinations performed and frequency:
- Clinical signs: All animals were observed for general condition and clinical signs at least once
daily throughout the study. All animals were observed for moribundity and mortality twice daily.

- Observation of detailed clinical signs: All animals were observed prior to dosing and once weekly for the dosing and
recovery periods.
The following items were observed:
-- Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion
-- Sutonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.)
-- Canges in gait, posture and response to handling, and the presence of clonic or tonic movements
-- Stereotypes (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walking backward, etc.)

- Sensory function and motor activity:
Six males and six females were randomly selected from the main groups in addition to all recovery animals and evaluated a few days before necropsy for
pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test and motor activity.

- Urinalysis:
Six males per group were randomly selected from the main group animals in addition to all male recovery animals for urinalysis two days before necropsy.
Fresh (3-hour) urine and 24-hour urine samples were collected from selected animals and analyzed.
Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
The following parameters were measured: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment, urine volume mL, specific gravity.

- Hematology:
Blood samples were taken from all males and all females from the main groups and all animals from the recovery groups.
All animals were fasted for approximately 18 hours prior to necropsy. At necropsy, all animals were anesthetized with isoflurane
and blood samples were collected from the abdominal aorta.
The following haematological parameters were examined:
Erythrocyte count (RBC), Hemoglobin (HGB), Flow cytometry, Cyanmethemoglobin 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:
Blood samples were collected from the abdominal aorta.
The following parameters were analyzed for all males and all females from the main groups and all animals from the recovery groups:
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Blood urea nitrogen (BUN), Urease-GLDH,
Creatinine (Crea), Total bilirubin (T-Bili), Vanadate oxidation, Total protein (TP), Albumin (Alb), Globulin (Glo), A/G ratio, Total cholesterol (T-Chol), Triglycerides (TG), Glucose (Glu), Calcium (Ca), Potassium (K), Ion-Sodium (Na), Chloride (Cl).

- Thyroid hormone analysis:
Blood samples were taken based on the following schedule:
- from all adult males and dams at termination
Animals were fasted for more than 18 hours before necropsy and anesthetized with isoflurane. Blood samples were collected from the abdominal aorta.
Thyroid hormones (T4 and TSH) were preferably measured as “total”.
The following parameters were analyzed: Total thyroxine (T4), Thyroid stimulating hormone (TSH).



Sacrifice and pathology:
- Necropsy:
-- The males of the main group were sacrificed on Day 50, and females of the main group were sacrificed on Postpartum Day 14.
-- All animals of the recovery groups were sacrificed two weeks after the final dosing.
-- All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia.
-- Complete gross postmortem examinations were conducted on all animals including the external surface and internal organs. All grossly visible
abnormalities were recorded.

- 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 following organs were weighed from all males and females from the main groups and recovery groups: Brain, Heart, Liver, Thymus, Spleen, Thyroid,
Adrenal, Kidney, Uterus, Ovary, Testis, Epididymis, Prostate + seminal vesicle with coagulating gland.

- Tissue preservation and slide preparation:
- All males and all females from the main groups in addition to all animals of the recovery groups were selected for tissue preservation.

- Histopathology:
Histopathological examinations were conducted as follows:
-- At least six males and six females from the control, low, mid and high groups (especially focused on spermatogenesis and interstitial testicular cell structure)
-- All tissues from animals found dead or killed in a moribund condition during the study.
-- All gross, macroscopic lesions
Statistics:
The statistical analysis of this study was conducted using the SAS program.
For the data including body weights, food consumption, thyroid hormone value, urine volume, hematology and
clinical chemistry parameters, organ weights, grip strength, motor activity, the Bartlett test was conducted to analyze for homogeneity of variance
(significance level: 0.05).
One-way analysis of variance (ANOVA) test was applied on homogeneous data, if significant (significance level: 0.05),
the Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The Kruskal-Wallis test was applied on heterogeneous data, then if significant (significance level: 0.05),
the Steel’s test was performed for multiple comparisons (significance levels: 0.05 and 0.01, two tailed).
The data of sensory function were analyzed utilizing the Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery group, the Folded-F test was applied to analyze homogeneity of variance (significance level: 0.05, two-tailed).
The Student t-test was applied for homogeneous data, but if overruled, the Aspin-Welch t-test was applied (significance
levels: 0.05 and 0.01, two-tailed).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, salivation was observed in five males and three females at 500 mg/kg/day.
In the recovery groups, salivation was temporarily observed in one male and one female at 500 mg/kg/day.
However, salivation was considered to have little toxicological significance since it was
caused by physicochemical characteristics.
In the main group, hematuria was observed in one female at 500 mg/kg/day from Post-partum Day (PPD) 6 to the end of dosing.
It was considered to be incidental.

No clinical signs were observed in males and females of the main groups and the recovery groups in the detailed examinations as carried out once a week.
Mortality:
no mortality observed
Description (incidence):
All animals of the main and the recovery groups survived the duration of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight changes were noted in males and females of
the main and the recovery groups when compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the main groups, statistically significant decreases in food consumption were temporarily noted in males at 50 and 150 mg/kg/day on Day 36
and/or Day 43.
A statistically significant increase in food consumption was temporarily noted in females at 500 mg/kg/day on Gestation Day (GD) 14.

However, these statistical significances in food consumption decreases had little toxicological relevance since they were not related to any body weight changes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No effects were observed in any animal in the main and the recovery groups.
Other statistical significances were considered not to be test substance-related changes
because of a small difference and/or the values were within the range of historical
reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed in any animal in the main and the recovery groups.
Other statistical significances were considered not to be test substance-related changes
because of a small difference and/or the values were within the range of historical
reference data.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the main groups, occult blood and erythrocytes were observed in the urine in two males at 500 mg/kg/day.
These two males also showed an increase in the amount of urine (about 5-15 % vs. 500 mg/kg/day group).
Two male animals at 500 mg/kg/day in the recovery group were noted with occult blood and showed an increase in the amount of urine (about 26-
28 % vs. 500 mg/kg/day group).

These findings were considered transient but test substance-related as these findings
were not found in any animal of the mid or low dose group.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and
corneal reflex test were observed in animals of both sexes in the main and the recovery
groups when compared to the control group.

In the main and the recovery groups, there were no test substance-related effects in the
grip strength test when compared to the control group.

In the main and the recovery groups, there was some statistical significance in the
spontaneous motor activity. However, the statistical significance had little toxicological
relevance because it was of small difference or temporary change and showed no dose dependency.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related adverse effects on organ weights were observed in any animal
in the main and the recovery groups.
Other statistical significances in the absolute and/or relative organ weights were
considered not to be test substance-related effects because of a small difference and/or
the values were within the range of historical reference data.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One female at 50 mg/kg/day showed a stress-related gross finding such as a small thymus.
It was considered not to be related to the test substance since a dose-response relationship was not observed.

The other macroscopic findings in the adult animals were considered to be incidental
and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related changes were not observed in this study.

A moderate cortical atrophy of the thymus was noted in one female at 50 mg/kg/day. It was considered not to be
related to the test substance since a dose-dependent response was not clear, and this non-specific finding was frequently noted
in animals of poor condition under stress.

In addition, occult blood and erythrocytes in urine were detected in two males at 500 mg/kg/day, and they were correlated with pyelitis and chronic
progressive nephropathy, respectively.
This finding was considered incidental and of no toxicological significance since there were no common microscopical findings related to
test substance at 500 mg/kg/day.

All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
There were no test substance-related adverse effects in total thyroxine (T4) and TSH
levels in adult males of the main and the recovery groups.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Key result
Critical effects observed:
no
Conclusions:
The NOAEL of Frambinon Methyl Ether for systemic toxicity was considered to be 500 mg/kg/day for the animals of both sexes.
Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of the substance on male and females Sprague-Dawley rats after repeated dose administration by oral gavage with dose levels of 50, 150, and 500 mg/kg body weight/day.

The animals of both sexes in the main and the recovery groups all survived the duration of the study.

No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights and thyroid hormone analysis in adult animals of both sexes in the test substance-dosed groups.

Salivation was observed in males and females at 500 mg/kg/day in the main and the recovery groups during the dosing period, but it was not considered to have toxicological significance since it was caused by physiochemical characteristics. Small thymus (cortical atrophy) was observed in one female at 50 mg/kg/day, it was considered not to be a test substance-related effect since there was no dose-dependent relationship. Occult blood and erythrocytes in urine were detected in two males at 500 mg/kg/day of the main group, and they were correlated with pyelitis and chronic progressive nephropathy, respectively. This finding was considered incidental and of no toxicological significance since there were no common microscopical findings related to test substance at 500 mg/kg/day. Still, two more male animals at 500 mg/kg/day in the recovery group were noted with occult blood.

These four males showed an increase in the amount of urine (about 5 % up to 28 % vs. 500 mg/kg/day group). Thus, these findings were considered transient but test item-related as these findings were not found in any animal of the mid or low dose group.

In conclusion, the NOAEL of Frambinon Methyl Ether for systemic toxicity was considered to be 500 mg/kg/day for the animals of both sexes.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
Based on a previously conducted 2-week repeated oral dose range finding study.

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(4-methoxyphenyl)butan-2-one
EC Number:
203-184-2
EC Name:
4-(4-methoxyphenyl)butan-2-one
Cas Number:
104-20-1
Molecular formula:
C11H14O2
IUPAC Name:
4-(4-methoxyphenyl)butan-2-one

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Specific Pathogen Free
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc., 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
- Age at the start of administrationn: 10 wks (males), 12 wks (females)
- Weight at the start of administration: Males: 353.4–406.6 g, Females: 216.7–288.1 g
- Fasting period before study: not specified
- Housing: Stainless wire mesh cages, 260W×350D×210H (mm); Polycarbonate cage, 260W×420D×180H (mm)
- Number of animals per cage: two animals (during quarantine-acclimation period), one animal (during dosing period),
one male and one female (during mating period), one female and neonates (during lactation period)
- Diet (e.g. ad libitum): ad libitum; pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water (e.g. ad libitum): ad libitum; public tap water, filtered and irradiated by ultraviolet light
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1–24.4°C
- Humidity (%): 39.0–60.7%
- Air changes (per hr): 10–15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Following the quarantine-acclimation period, vaginal smears were prepared daily
for females in order to examine the estrous cycle for 2 weeks before group
assignment. Healthy 60 males and 60 females showing normal estrous cycles were
selected and distributed into four groups of 12 males and 12 females per group for
the main study as well as additional 6 animals of each sex in the control and high
dose groups for a 2-week recovery period.
Animals were randomly assigned to groups in an attempt to equalize mean group body weights.
A temporary identification number was marked on the tail using a red indelible pen.

Individual doses were calculated for each animal based on the most recently
recorded body weight data at a dose volume of 2 mL/kg. Dosing solutions were
administered once daily using 1 or 3 mL disposable syringes fitted with a gastric
intubation tube.

Males of the main group were dosed once daily for a total of 49 days (for 2 weeks
prior to mating, during 2 weeks of mating and 21 days of post-mating). Also, males
and females of the recovery groups were dosed once daily for 49 days.
Females of the main group were dosed once daily for 2 weeks prior to mating until
Postpartum Day 13. Also, females showing no evidence of parturition signs were
dosed until Gestation Day 25.

The control group animals were dosed with the vehicle only with the same dose
volume as the test substance-treated groups.


Details on mating procedure:
Rats of the same group were mated at a ratio of 1:1 for a period of two weeks.
All females were examined for the presence of a vaginal plug or sperm in the
vaginal smear twice a day (AM and PM) for the confirmation of mating. The
day of positive confirmation was defined as Gestation Day 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the treatment period, formulation samples were prepared and analysed for stability and
homogeneity of the test item in the selected vehicle.
Verification of dose level concentrations were conducted using Gas Chromatography
Duration of treatment / exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2
weeks of mating and 21 days of post-mating), and females of the main group were dosed once
daily for 2 weeks prior to mating, throughout gestation and for 13 days after delivery.
Males and females of the recovery groups were dosed once daily for 49 days.
Females showing no evidence of parturition signs were dosed until Gestation Day 25
Frequency of treatment:
7 days per week
Details on study schedule:
All animals were observed for general condition and clinical signs at least once
daily throughout the study.
All animals were observed for moribundity and mortality twice daily.
Females were also observed for signs of abortion and premature birth.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
conntrol group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
- 12 animals per sex per group
- additional 6 animals per sex per group for the recovery groups (control and high dose)
Control animals:
yes, concurrent vehicle
Details on study design:
Healthy 60 males and 60 females showing normal estrous cycles were selected and distributed into four groups of 12 males and 12 females
per group for the main study as well as additional 6 animals of each sex in the control and high dose groups for a 2-week recovery period.
Animals were randomly assigned to groups in an attempt to equalize mean group body weights.
All animals were examined for general health upon receipt.
Body weights were recorded on the days of receipt, the last day of the quarantine-acclimation period
and the day of group assignment.
All animals were quarantined and acclimated for 7 days and observed once daily for general health
condition and clinical signs.
After the quarantine-acclimation period, all animals were observed once daily for clinical signs until
group assignment.
Following group assignment, animals were uniquely identified by a blue indelible marking on the tail. A
color-coded cage card was placed on each animal’s cage displaying the group and dose level information.

- Dose selection rationale:
According to the results of a previous dose range finding study the doses were selected for the 3 dose
groups.

- Body weights:
-- Body weights of males of the main group and animals of both sexes of the recovery groups were re
corded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and
recovery periods, the day prior to 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 (the first day of dosing),
once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to
necropsy and on the day of necropsy.
Fasted body weights recorded on the day of necropsy were presented, but were not included in the
statistical analysis.

- Food consumption:
-- Food consumption of males of the main group and animals of both sexes of the recovery group was
recorded just prior to dosing on Day 0 (one day before first
dosing), once a week during the dosing and recovery periods (except during mating) and the day prior
to necropsy.
-- Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before first dosing),
once a week throughout the dosing period (except during mating), on Gestation Days 0, 6, 13 and 19, on Postpartum Days 0, 3
and 12 and the day prior to necropsy. Residual feed was recorded on the next day.
Food consumption was not recorded during mating.
Individual food consumption was calculated by subtracting the amount of residual feed from the
amount presented.

The males of the main group were sacrificed on Day 50, and females of the main group were sacrificed on Postpartum Day 14.
All animals of the recovery groups were sacrificed two weeks after the final dosing. Non-pregnant females were sacrificed on Gestation Day 26.
Dams, whose pups were all dead, were sacrificed as soon as possible.
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
All animals were observed for general condition and clinical signs at least once
daily throughout the study. All animals were observed for moribundity and
mortality twice daily. Females were also observed for signs of abortion and
premature birth.

- Observation of detailed clinical signs: All animals were observed prior to dosing and once weekly for
the dosing and recovery periods.
The following items were observed:
-- Skin, fur, eyes, mucous membranes, occurrence of secretion and excretion
-- Sutonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern, etc.)
-- Canges in gait, posture and response to handling, and the presence of clonic or tonic movements
-- Stereotypes (excessive grooming, repetitive circling, etc.) or bizarre behavior (self-mutilation, walk
ing backward, etc.)

- Sensory function and motor activity:
Six males and six females were randomly selected from the main groups in addition
to all recovery animals and evaluated for the followings a few days before necropsy.
The tests on the dams of the main group were carried out on lactation days 6–11:
pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test and motor a
ctivity.

- Urinalysis:
Six males per group were randomly selected from the main group animals in addition to all male
recovery animals for urinalysis two days before necropsy.
Fresh (3-hour) urine and 24-hour urine samples were collected from selected animals and analyzed.
Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
The following parameters were measured: pH, protein, glucose, bilirubin, occult blood, color and t
urbidity, sediment, urine volume mL, specific gravity.

- Hematology:
Blood samples were taken from all males and all females from the main groups and all animals from
the recovery groups.
All animals were fasted for approximately 18 hours prior to necropsy. At necropsy, all animals were
anesthetized with isoflurane
and blood samples were collected from the abdominal aorta.
The following haematological parameters were examined:
Erythrocyte count (RBC), Hemoglobin (HGB), Flow cytometry, Cyanmethemoglobin Hematocrit
(HCT), Mean corpuscular volume (MCV),
Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Pla
telets (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:
Blood samples were collected from the abdominal aorta.
The following parameters were analyzed for all males and all females from the main groups and all
animals from the recovery groups:
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP),
Blood urea nitrogen (BUN), Urease-GLDH,
Creatinine (Crea), Total bilirubin (T-Bili), Vanadate oxidation, Total protein (TP), Albumin (Alb),
Globulin (Glo), A/G ratio, Total cholesterol (T-Chol), Triglycerides (TG), Glucose (Glu), Calcium (Ca),
Potassium (K), Ion-Sodium (Na), Chloride (Cl).

- Thyroid hormone analysis:
Blood samples were taken based on the following schedule:
-- from at least two pups (preferably one male and one female) per litter on PNDs 4 and on PND 13, each
(When there was a sufficient number of pups, blood samples were taken from the pups available above the culling target of 8 pups/litter, normally from at least
two pups per litter on both PNDs 4 and 13, otherwise.
When only one pup was available above the culling target, only one pup was used for the determination of PND 4. When the litter size was below the culling
target, blood collection was not carried out on PND 4. On PND 13, blood samples of all male and female pups were taken and pooled according to sex for
analysis.)
-- from all adult males and dams at termination
Animals were fasted for more than 18 hours before necropsy (except pups) and anesthetized with isoflurane.
Blood samples were collected from the heart (PND 4) or abdominal aorta. Blood samples of male and female pups were pooled by the litter.
Serum samples from the pups on PND 13 and all adult males were analyzed.
Thyroid hormones (T4 and TSH) were preferably measured as “total”.
The following parameters were analyzed: Total thyroxine (T4), Thyroid stimulating hormone (TSH).
Oestrous cyclicity (parental animals):
The estrous cycle examination for females of the main group was conducted from the dosing initiation day
to the confirmed copulation day and necropsy day.
Smears of vaginal mucosa were prepared daily in the morning.

Vaginal smears were examined on the day of necropsy to determine the stage of the
estrous cycle and allowed correlation with histopathology of the female reproductive organs.
Sperm parameters (parental animals):
Histopathological examinations of at least six males and six females from the control, low, mid and high groups focused on spermatogenesis and interstitial testicular cell structure.
Litter observations:
Pups were observed for mortality once daily. The presence of dead pups was observed on Postnatal Days (PND) 0 and 4.
The results were calculated as follows:
- Mean litter size
- Live birth index (%) = (Number of live pups on PND 0 / Number of implantations) × 100
- Viability index on postnatal day 0 (%) = (Number of pups born alive on PND 0 / Total number of pups born) × 100
- Viability index on postnatal day 4 (%) = (Number of pups surviving on PND 4 / Number of pups born alive on PND 0) × 100

External examination and determination of sex ratio of pups:
External examination of all pups was conducted and sex ratio of all pups was determined on delivery day and on PNDs 4 and 13.
All grossly visible abnormalities were recorded. Pups were euthanized after external examination on PNDs 4 and 13.
- Sex ratio = Total number of live male pups / Total number of live female pups (PND 0)

Body weights of pups:
Body weights of pups were recorded on PNDs 0, 4 and 13.

Anogenital distance (AGD)
AGD of each pup was measured on PND 4 using a digimatic caliper (Mitutoyo,
Japan). AGD index was calculated as follows:
- AGD index = AGD /3√ body weight

Culling and individual identification of pups:
Four male and four female pups were randomly selected from each litter on PND 4 for culling. Extra pups were euthanized.
Litters of 8 pups or less were not culled. When litters contained less than four pups of one sex, pups of the opposite sex were added to make a total of 8 pups.
Individual identification of pups was conducted on PND 4 after culling.

Nipple retention:
The number of nipples of male pups was counted on PND 12.
Postmortem examinations (parental animals):
- Necropsy:
-- The males of the main group were sacrificed on Day 50, and females of the main group were sacrificed on Postpartum Day 14.
-- All animals of the recovery groups were sacrificed two weeks after the final dosing.
-- All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane
anesthesia.
-- Complete gross postmortem examinations were conducted on all animals including the external
surface and internal organs.
All grossly visible abnormalities were recorded.

- 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 following organs were weighed from all males and females from the main groups and recovery
groups: Brain, Heart, Liver, Thymus, Spleen, Thyroid,
Adrenal, Kidney, Uterus, Ovary, Testis, Epididymis, Prostate + seminal vesicle with coagulating gland.

- Tissue preservation and slide preparation:
All males and all females from the main groups in addition to all animals of the recovery groups were selected for tissue preservation.

- Histopathology:
Histopathological examinations were conducted as follows:
-- At least six males and six females from the control, low, mid and high groups (especially focused on spermatogenesis and interstitial testicular cell structure)
-- All tissues from animals found dead or killed in a moribund condition during the study.
-- All gross, macroscopic lesions
Postmortem examinations (offspring):
Dead pups and pups sacrificed on PND 13 or shortly thereafter, were carefully
examined externally for gross abnormalities. Attention was paid to the external
reproductive genitals which should be examined for signs of altered development.

Number of uterine implantations:
The uterine implantation sites were counted at necropsy. Post-implantation loss rate was calculated as follows:
- Post-implantation loss rate (%) = ((Number of implantations – Number of live pups) / Number of implantations) × 100

All surviving animals were sacrificed and complete gross postmortem examinations were conducted on all
animals including the external surface and internal organs. All grossly visible abnormalities were recorded.
Statistics:
The statistical analysis of this study was conducted using the SAS program.
For the data including body weights, food consumption, estrous cycle, mating period,
gestation period, post-implantation loss, body weights and birth and survival rates of
pups, AGD index, nipple number, thyroid hormone value, urine volume, hematology and
clinical chemistry parameters, organ weights, grip strength, motor activity, the Bartlett
test was conducted to analyze for homogeneity of variance (significance level: 0.05).
One-way analysis of variance (ANOVA) test was applied on homogeneous data, then if
significant (significance level: 0.05), the Dunnett’s t-test was applied for multiple
comparisons (significance levels: 0.05 and 0.01, two-tailed).
The Kruskal-Wallis test was applied on heterogeneous data, then if significant (significance level: 0.05), the Steel’s
test was performed for multiple comparisons (significance levels: 0.05 and 0.01, two tailed).
The data of sensory function, mating index, fertility index and other data associated with
gestation were analyzed utilizing the Fisher’s exact test (significance levels: 0.05 and 0.01).
For the data of recovery group, the Folded-F test was applied to analyze homogeneity of
variance (significance level: 0.05, two-tailed).
The Student t-test was applied for homogeneous data, but if overruled, the Aspin-Welch t-test was applied (significance
levels: 0.05 and 0.01, two-tailed).
Reproductive indices:
Delivery observation:
Animals not showing parturition signs until Gestation Day 25 were evaluated
as non-pregnant animals. Final pregnancy diagnosis was decided by
observation of uterine implantation sites at necropsy.
When dams delivered between 9:00 AM and 4:30 PM, that day was defined as Postpartum Day 0.
When they delivered after 4:30 PM, the following day was recorded as Postpartum Day 0.
The mating, gestation and parturition were observed. The following reproduction indexes were calculated:
- Mating index (%) = (Number of females with confirmed mating / Number of females placed with males) × 100
- Mating period = Day of confirmed mating – Day of initial mating (based on the dosing day)
- Gestation period = Postpartum Day 0 – Gestation Day 0 (based on the dosing day)
- Male fertility index (%) = (Number of males impregnating a female / Number of males with confirmed mating) ×100
- Female fertility index (%) = (Number of pregnant females / Number of females with confirmed mating) × 100
- Gestation index (%) = (Number of females with live pups / Number of pregnant females) × 100

Number of uterine implantations:
The uterine implantation sites were counted at necropsy. Post-implantation loss rate was calculated as follows:
- Post-implantation loss rate (%) = ((Number of implantations – Number of live pups) / Number of implantations) × 100
Offspring viability indices:
Pups were observed for mortality once daily. The presence of dead pups was observed on Postnatal Days (PND) 0 and 4.
The results were calculated as follows:
- Mean litter size
- Live birth index (%) = (Number of live pups on PND 0 / Number of implantations) × 100
- Viability index on postnatal day 0 (%) = (Number of pups born alive on PND 0 / Total number of pups born) × 100
- Viability index on postnatal day 4 (%) = (Number of pups surviving on PND 4 / Number of pups born alive on PND 0) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In the main groups, salivation was observed in five males and three females at 500 mg/kg/day.
In the recovery groups, salivation was temporarily observed in one male and one female at 500 mg/kg/day.
However, salivation was considered to have little toxicological significance since it was
caused by physicochemical characteristics.
In the main group, hematuria was observed in one female at 500 mg/kg/day from Post-partum Day
(PPD) 6 to the end of dosing.
It was considered to be incidental.

No clinical signs were observed in males and females of the main groups and the recovery groups in
the detailed examinations as carried out once a week.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals of the main and the recovery groups survived the duration of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight changes were noted in males and females of
the main and the recovery groups when compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the main groups, statistically significant decreases in food consumption were temporarily noted in
males at 50 and 150 mg/kg/day on Day 36.
A statistically significant increase in food consumption was temporarily noted in females at 500 mg/kg/day on Gestation Day (GD) 14.

However, these statistical significances in food consumption decreases had little toxicological
relevance since they were not related to any body weight changes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No effects were observed in any animal in the main and the recovery groups.
Other statistical significances were considered not to be test substance-related changes
because of a small difference and/or the values were within the range of historical
reference data.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse effects were observed in any animal in the main and the recovery groups.
Other statistical significances were considered not to be test substance-related changes
because of a small difference and/or the values were within the range of historical
reference data.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In the main groups, occult blood and erythrocytes were observed in the urine in two males at 500 mg/kg/day.
These two males also showed an increase in the amount of urine (about 5-15 % vs. 500 mg/kg/day group).
Two male animals at 500 mg/kg/day in the recovery group were noted with occult blood and showed
an increase in the amount of urine (about 26-28 % vs. 500 mg/kg/day group).

These findings were considered transient but test substance-related as these findings
were not found in any animal of the mid or low dose group.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and
corneal reflex test were observed in animals of both sexes in the main and the recovery
groups when compared to the control group.
In the main and the recovery groups, there were no test substance-related effects in the
grip strength test when compared to the control group.
In the main and the recovery groups, there was some statistical significance in the
spontaneous motor activity.
However, the statistical significance had little toxicological relevance because it was of small difference or temporary change and showed no dose dependency.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related changes were not observed in this study.
A moderate cortical atrophy of the thymus was noted in one female at 50 mg/kg/day. It was considered not to be
related to the test substance since a dose-dependent response was not clear, and this non-specific finding was frequently noted
in animals of poor condition under stress.
In addition, occult blood and erythrocytes in urine were detected in two males at 500 mg/kg/day, and they were correlated with pyelitis and chronic
progressive nephropathy, respectively.
This finding was considered incidental and of no toxicological significance since there were no common microscopical findings related to
test substance at 500 mg/kg/day.
All microscopic findings seen in other organs and tissues were considered to be incidental and of no toxicological significance.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis:
There were no test substance-related adverse effects in total thyroxine (T4) and TSH
levels in adult males of the main and the recovery groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle lengths (day) of females in the 0 (control), 50, 150 and 500 mg/kg/day
dose groups were all 4.0 days. There were no statistically significant differences in any
dosing group.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating and Gestation:
In the control group and 50, 150 and 500 mg/kg/day dose groups, the mating periods
were 3.1, 2.3, 2.4 and 2.8 days, respectively, the mating index was all 100%, the
gestation periods were 22.0, 22.2, 22.2 and 22.3 days, respectively, and the fertility
indices of animals of both sexes were 100.0, 91.7, 100.0 and 100.0%, respectively.
There were no statistically significant differences in any dosing group.

Delivery and Pups Examinations:
In the control group and 50, 150 and 500 mg/kg/day dose groups, the gestation
indices were 100.0, 90.9, 100.0 and 100.0%, the post-implantation loss rates were 8.9,
12.7, 10.2 and 9.6%, the live birth indices were 91.1, 87.4, 89.8 and 90.4%, the mean
litter sizes were 13.6, 14.9, 13.1 and 14.2, the viability indices on Postnatal Day
(PND) 0 were 99.5, 89.1, 98.5 and 95.2, and the viability indices on PND 4 were
98.2, 97.9, 98.9 and 95.8%, respectively.
In addition, stillbirth was observed in one female at 50 mg/kg/day. The sex ratios on
PND 0 were 1.0, 1.2, 0.8 and 0.9, respectively.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
External examinations of pups:
There were no test substance-related changes in the external findings in pups at 50,
150, and 500 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
In the control group and 50, 150 and 500 mg/kg/day dose groups, the gestation
indices were 100.0, 90.9, 100.0 and 100.0%, the post-implantation loss rates were 8.9,
12.7, 10.2 and 9.6%, the live birth indices were 91.1, 87.4, 89.8 and 90.4%, the mean
litter sizes were 13.6, 14.9, 13.1 and 14.2, the viability indices on Postnatal Day
(PND) 0 were 99.5, 89.1, 98.5 and 95.2, and the viability indices on PND 4 were
98.2, 97.9, 98.9 and 95.8%, respectively.
In addition, stillbirth was observed in one female at 50 mg/kg/day. The sex ratios on
PND 0 were 1.0, 1.2, 0.8 and 0.9, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the body weights of pups at 50, 150 and
500 mg/kg/day on PNDs 0, 4 and 13.
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 specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy:
No test substance-related macroscopic findings were observed in live and dead F1 pups
of both sexes.
Histopathological findings:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance (AGD) of pups:
There were no test substance-related changes in the AGD indexes of pups at 50, 150
and 500 mg/kg/day on PND 4.

Nipple retention of F1 males:
The nipple number was 0 in male pups at 0, 50, 150 and 500 mg/kg/day on PND 12.
There was no nipple retention in male pups at 50, 150 and 500 mg/kg/day on PND 12.

Thyroid Hormone Analysis:
There were no test substance-related adverse effects in total thyroxine (T4) and TSH
levels in F1 pups on PND 13 at 50, 150 and 500 mg/kg/day.

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:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive and developmental toxicity screening

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL of the test item for reproductive toxicity was considered to be 500 mg/kg/day for males and females.
The NOAEL for developmental toxicity was considered to be 500 mg/kg/day for pups.
Executive summary:

The purpose of this study was to determine the No Observed Adverse Effect Level (NOAEL) of the test item for the reproduction/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential of the test substance when administered orally to male and female rats at dose levels of 0 (control), 50, 150 and 500 mg/kg/day.

Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating), and females of the main group were dosed once daily for 2 weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery groups were dosed for 49 days. The study was performed according to OECD TG 422 and in compliance to GLP.

The animals of both sexes in the main and the recovery groups all survived the duration of the study. No test substance-related adverse effects were noted in the results of body weights, food consumption, sensory function, motor activity, urinalysis, hematology, clinical chemistry, organ weights and thyroid hormone analysis in adult animals of both sexes in the test substance-dosed groups.

Stillbirth was observed in one female at 50 mg/kg/day. It was considered to be incidental since there was no dose-dependency.

Small thymus (cortical atrophy) was observed in one female at 50 mg/kg/day that showed stillbirth, however, it was considered not to be a test substance-related effect since there was no dose-dependent relationship.

No test substance-related adverse effects were noted in the results of the estrous cycle, mating period, mating index, gestation period, male and female fertility indexes, gestation index, post-implantation loss rate, live birth index, mean litter size, external examination of pups, body weight of pups, sex ratio of pups and viability indexes of Postnatal Days 0 and 4. No test substance-related effects were noted in the results of anogenital distance (AGD) index of pups, nipple retention of male pups and T4 and TSH of pups.

Therefore, the test item had no endocrine disrupting potential in the pups under the condition of this study.

Based on these results of this study, the NOAEL for reproductive toxicity was considered to be 500 mg/kg/day for males and females. The NOAEL for developmental toxicity was considered to be 500 mg/kg/day for pups.