Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2019-07-11 to 2019-12-25
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:
From 2019-07-11 to 2019-12-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
Three females were dosed twice a day on day 39 since these animals were administered before measurements of their body weights on this day. Total of dosing volume was correct and did not affect evaluation of the study results.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Batch No.: 707001
Purity: 98.6%
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
This strain is established as a laboratory animal and has historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Hino Breeding Center
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 340.0-391.1 g and 220.6-264.3g for males and females, respectively.
- Housing: Housed in hanging stainless steel cages with wire-mesh floor (260 Wx380 Dx180 H mm, TOKIWA) for quarantine and acclimation. Males and females were housed in 260 Wx380 Dx180 H mm and 165 Wx300 Dx150H mm of hanging stainless cages with wire mesh floor (TOKIWA) individually after group allocation, respectively with irradiated hemp mats with gamma-ray. In the mating period each female was housed in a male's cage in each dose group. Copulated females were housed in polycarbonate cages (PC cage, 265 Wx426 Dx150 H mm, TOKIWA) with autoclaved wooden bedding, and enrichments of autoclaved gnawing wood and hemp mats from gestation day 14. Pups were housed with a dam after delivery.
- Diet: pelleted diet, ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
The analytical data of contaminants in the diet were confirmed to meet the requirements of the “Toxic Substances Control Act of US-EPA" (1979). Contaminants in drinking water were analyzed twice a year according to the water regulations of the “Ordinance on drinking water quality standards" [No. 101 of MHLW]. The analytical data of contaminants in the water were confirmed to be in the stated ranges of the lab.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours light (7:00-19:00): 12 hour dark(19:00-7:00).
Route of administration:
oral: gavage
Details on route of administration:
The vehicle and formulations were administered by gavage once a day using a syringe (Terumo) connected to a nelaton catheter (Terumo) at the amount of 4 mL/kg based on the latest body weights.
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed, and olive oil was added to prepare the 7.50 w/v% formulation. A part of the 7.50 w/v% formulation was taken and diluted with olive oil to prepare the 0.750 and 2.50 w/v% formulation.
The formulations and vehicle were subdivided into plastic containers and stored at a cold place (target range: 1 to 10°C). On each dosing day, formulations and vehicle were taken out from the storage place and dosed to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Since solubility to water was 7.26 mg/L, olive oil was used for a preparations investigation of the dosing formulations. The test item dissolved in olive oil at concentration of 25 w/v% (non-GLP).
This vehicle is used for a general toxicity study and historical control data existed.
- Concentration in vehicle: 7.50, 2.50, 0.750 w/v%
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): 801029
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item formulations were confirmed with GC in the first preparation.
Actual concentrations of the 7.50, 2.50 and 0.750 w/v% formulations were 7.34, 2.42 and 0.717 w/v%, respectively.
Duration of treatment / exposure:
Male and female rats were treated with test item for 14 days and set on mating period for maximum of 14 days. Males and females were administrated until day 29 of dosing and 13 days after delivery, respectively.
Frequency of treatment:
Once daily. Three females were treated twice a day on day 39.
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Intermediate dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
10 male/ female/ dose
non-mating satellite group: 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A 14-day repeated-dose oral toxicity study was performed at the dose levels of 0, 50, 200, 500 and 1000 mg/kg/day consisting three males and three females for each group. Salivation was observed in the 200, 500 and 1000 mg/kg groups. Decreased spontaneous locomotion, decreased respiratory rate and incomplete eyelids opening were observed in the 500 and 1000 mg/kg groups. Staining lower abdomen, restlessness and staggering gait were observed in the 1000 mg/kg group. A decrease or a trend toward decrease in body weights were noted in the 500 and 1000 mg/kg groups. In organ weights, absolute and relative weights of the liver were increased and absolute weight of the spleen was decreased in the 500 and 1000 mg/kg groups. Absolute weights of the testes were decreased and relative weights of the kidneys were increased in the 1000 mg/kg group. In necropsy, enlargement of the liver was observed in the 500 and 1000 mg/kg groups. Apparent spotty pattern of the surface of the kidneys was observed in males of the 500 mg/kg group. Small testes and thymus, elevation of the limiting ridge of the forestomach were observed in the 1000 mg/kg group. Therefore 300 mg/kg/day was set for the high dose, and 100 and 30 mg/kg/day were set for the middle and low doses for the present study.
- Fasting period before blood sampling for clinical biochemistry: 16-20 h
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing period: Twice a day; Recovery period: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the dosing period and once a week thereafter.

SENSORIMOTOR FUNCTION EXAMINATIONS: yes
- Time schedule: The animals were examined for the parameters of the following listed in five males of each dose groups, non-mating five females of the control and 300 mg/kg groups, and selected five parental females of each dose group in each last dosing week. Not performed for the recovery period.
- Reflex (response to optic, pinna and pain, pupillary reflex, air righting reflex) tests and measurements of grip strengths were performed on a blind test basis. Locomotor activity of each animal was counted.


BODY WEIGHT: Yes
- Time schedule for examinations:
Males and non-mating females: Days 1, 3, 7, 14, 21 and 28 of dosing, days 1, 7 and 14 of recovery, and necropsy day.
Mating females: Days 1, 3, 7 and 14 of dosing, gestation days 0, 7, 14 and 20, postnatal days 0, 4, 8 and 13, and necropsy day.
Dead animals were weighed on the day found dead. Non-coupled female was weighed once a week after day 21 and necropsy day (day 55, equivalent pregnant day 26).


FOOD CONSUMPTION: yes
- Mean food consumption per day was calculated from their feeding and remainder weights.
- Time schedule for examinations:
Males and non-mating females: Days 1, 3, 7, 14, 21 and 28 of dosing, days 1, 7 and 14 of recovery
Mating females: Days 1, 3, 7 and 14 of dosing, gestation days 0, 7, 14 and 20, postnatal days 0, 4, 8 and 13


WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 16-20 h of the start of fasting
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: five males, five mating females for each dose group and non-mating five females and males and females of the recovery group
- Whole blood or plasma samples were examined. Parameters examined see "Examination parameters" in attached background material.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 16-20 h of the start of fasting
- Animals fasted: Yes
- How many animals: five males, five mating females for each dose group and non-mating five females and males and females of the recovery group
- Serum samples were used, Parameters examined see "Examination parameters" in attached background material.

URINALYSIS: Yes
- Time schedule for collection of urine: at afternoon of the each last observation day
- Metabolism cages used for collection of urine: Yes, Five males of the main group (same animals of sensorimotor function examinations), non-mating females, and male and females of the recovery group were housed in a metabolic cages at afternoon of the each last observation day with free drinking and no feed until next morning for 15-17 h.
- Animals fasted: Yes
- Parameters examined see "Examination parameters" in attached background material.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see "Examination parameters" in attached background material)

HISTOPATHOLOGY: Yes (see "Examination parameters" in attached background material)
Statistics:
Data regarding body weights of parental animals, food consumption, grip strength, locomotor activity counts, parameters of the hematological and blood chemical examinations, urine volume, specific gravity of urine, organ weights, body weights on necropsy day were analyzed by the Barlett's test for homogeneity of variance. If significant difference (p<0.05) was not noted, the values were analyzed by the Dunnett's test. If significant difference (p<0.05) was noted in the Bartlett test, the values were analyzed by the nonparametric Dunnett’s test. The frequencies of defecation and urination during the dosing period were analyzed by the nonparametric Dunnett's test. Data regarding body weights and food consumption during the recovery period, and parameters of the hematological and blood chemical examinations, urine volume, specific gravity of urine, organ weights and body weights on the necropsy day for the recovery group were analyzed by the F-test for variance ratio. If there were no significant differences at a significance level of 5%, the Student's t-test was performed. If there were significant differences, the Aspin-Welch t-test was performed. The frequencies of defecation and urination during the recovery period were analyzed by the Mann-Whitney U-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed immediately after dosing to males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group. Soft stool was observed in males and non-mating females of the 300 mg/kg group. Slight decreased spontaneous locomotion and lacrimation were observed in females of the 300 mg/kg group on day 22 of gestation period. During the recovery period soft stool was observed in males of the 300 mg/kg group and disappeared by day 3.
Detailed clinical observations were not affected by the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 300 mg/kg group, one animal was died during delivery (day 23 of the gestation period) and other animal was died on day 22 of the gestation period.
These animals did not show any severe changes compared to survived animals, and they were considered to have increased-stress related to delivery since small thymus and enlargement of the adrenals were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were decreased in males of the 100 and 300 mg/kg groups. During the recovery period body weights were decreased in males of the 300 mg/kg group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was decreased in males of the 100 and 300 mg/kg groups and females of the 300 mg/kg 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):
Hemoglobin conc. was decreased in males of the 300 mg/kg group. Prothrombin time was elongated in non-mating females of the 300 mg/kg group. At the end of the recovery period reticulocytes were increased in males of the 300 mg/kg group.
The test item affected the blood coagulation system.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, ALP was decreased in the 30, 100 and 300 mg/kg groups. ALT was decreased in the 100 and 300 mg/kg groups. T4 was increased in the 100 mg/kg group without dose-relationship. Potassium was decreased in the 300 mg/kg group.
In mating and non-mating females, ALT was decreased in the 300mg/kg group.
These changes were considered to be related to the decreases in body weights and food consumption, no toxicologically significant
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In urinalyses, renal tubular epithelial cells were observed in males of the 30, 100 and 300 mg/kg groups. Cloudy urine was observed in males of the 300 mg/kg group.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Grip strength of forelimbs was decreased to mating females of the 300 mg/kg group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative weight of the liver was increased in males of the 100 and 300 mg/kg groups and non-mating females of the 300 mg/kg group. At the end of the recovery period relative weights of the kidneys were increased in males of the 300 mg/kg group.
The weight change of the liver in 100 and 300 mg/kg groups was decided to be toxicologically significant since no histopathological changes were observed in the lowest dose of 30 mg/kg group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Apparent spotty pattern of the surface of the kidneys was observed in males of the 30, 100 and 300 mg/kg groups. Enlargement of the liver was observed in males and females of the 300 mg/kg group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Accumulation of the hyaline droplets in the proximal tubules, increased eosinophilic bodies and regeneration of the tubules in the outer medulla of the kidney were observed in males of the 30, 100 and 300 mg/kg groups.
Centrilobular hypertrophy of the hepatocytes of the liver was observed in males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group. At the end of the recovery period granular cast and regeneration of the tubules in the outer medulla of the kidney were observed in males of the 300 mg/kg group.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

The changes in urinalyses, organ weights and histopathology related to the kidney were considered to be male rats specific and no toxicologically significant since the hyaline droplets were confirmed to be related to α2u-globuin.

Conclusions:
In conclusion, the No-Observed-Adverse-Effect Level (NOAEL) for repeated-dose toxicity under the conditions tested was considered to be 30 mg/kg/day due to decreases in body weights and food consumption, an increase in relative weight of the liver and centrilobular hypertrophy of the hepatocytes in the 100 and 300 m/kg groups.
Executive summary:

"A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test" was performed to assess the repeated-dose, reproductive and developmental toxicities of test item in accordance with OECD 422.

Male and female Crl:CD(SD) rats at 9 weeks of age was treated with the test item for 14 days and set on mating period for maximum of 14 days. Males and females were administrated until day 29 of dosing and 13 days after delivery, respectively. The dosage levels were set at 0 (olive oil), 30, 100 and 300 mg/kg/day. Recovery groups were set for the 0 and 300 mg/kg groups after 29 days treatment for males and females of the satellite group.

Salivation was observed immediately after dosing to males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group. Soft stool was observed in males and non-mating females of the 300 mg/kg group. Slight decreased spontaneous locomotion and lacrimation were observed in females of the 300 mg/kg group on day 22 of gestation period. One each female was died on days 22 and 23 of the gestation period. During the recovery period soft stool was observed in males of the 300 mg/kg group and disappeared by day 3.

In sensorimotor function examinations, grip strength of forelimbs was decreased to mating females of the 300 mg/kg group.

Body weights were decreased in males of the 100 and 300 mg/kg groups. During the recovery period body weights were decreased in males of the 300 mg/kg group.

Food consumption was decreased in males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group.

In urinalyses, renal tubular epithelial cells were observed in males of the 30, 100 and 300 mg/kg groups. Cloudy urine was observed in males of the 300 mg/kg group.

In hematology, hemoglobin conc. was decreased in males of the 300 mg/kg group. Prothrombin time was elongated in non-mating females of the 300 mg/kg group. At the end of the recovery period reticulocytes were increased in males of the 300 mg/kg group.

In organ weights, relative weight of the liver was increased in males of the 100 and 300 mg/kg groups and non-mating females of the 300 mg/kg group. At the end of the recovery period relative weights of the kidneys were increased in males of the 300 mg/kg group.

In necropsy, apparent spotty pattern of the surface of the kidneys was observed in males of the 30, 100 and 300 mg/kg groups. Enlargement of the liver was observed in males and females of the 300 mg/kg group.

In histopathological examinations, accumulation of the hyaline droplets in the proximal tubules, increased eosinophilic bodies and regeneration of the tubules in the outer medulla of the kidney were observed in males of the 30, 100 and 300 mg/kg groups.The changes in urinalyses, organ weights and histopathology related to the kidney were considered to be no toxicologically significant since the hyaline droplets were confirmed to be related toα2u-globuin.Centrilobular hypertrophy of the hepatocytes of the liver was observed in males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group. At the end of the recovery period granular cast and regeneration of the tubules in the outer medulla of the kidney were observed in males of the 300 mg/kg group.

Detailed clinical observations and blood chemistry were not affected by the test item.

In conclusion, the No-Observed-Adverse-Effect Level (NOAEL) for repeated-dose toxicity under the conditions tested was considered to be 30 mg/kg/day due to decreases in body weights and food consumption, an increase in relative weight of the liver and centrilobular hypertrophy of the hepatocytes in the 100 and 300 m/kg groups.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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:
2016
Deviations:
yes
Remarks:
Three females were dosed twice a day on day 39 since these animals were administered before measurements of their body weights on this day. Total of dosing volume was correct and did not affect evaluation of the study results.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetravinylsilane
EC Number:
214-192-0
EC Name:
Tetravinylsilane
Cas Number:
1112-55-6
Molecular formula:
C8H12Si
IUPAC Name:
tetravinylsilane
Test material form:
liquid
Specific details on test material used for the study:
Batch No.: 707001
Purity: 98.6%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
This strain is established as a laboratory animal and has historical data.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Charles River Japan Hino Breeding Center
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 340.0-391.1 g and 220.6-264.3g for males and females, respectively.
- Housing:
Housed in hanging stainless steel cages with wire-mesh floor (260 Wx380 Dx180 H mm, TOKIWA) for quarantine and acclimation. Males and females were housed in 260 Wx380 Dx180 H mm and 165 Wx300 Dx150H mm of hanging stainless cages with wire mesh floor (TOKIWA) individually after group allocation, respectively with irradiated hemp mats with gamma-ray. In the mating period each female was housed in a male's cage in each dose group.
- Diet: pelleted diet, ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours light (7:00-19:00): 12 hour dark(19:00-7:00).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed, and olive oil was added to prepare the 7.50 w/v% formulation. A part of the 7.50 w/v% formulation was taken and diluted with olive oil to prepare the 0.750 and 2.50 w/v% formulation.
The formulations and vehicle were subdivided into plastic containers and stored at a cold place (target range: 1 to 10°C). On each dosing day, formulations and vehicle were taken out from the storage place and dosed to the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Since solubility to water was 7.26 mg/L, olive oil was used for a preparations investigation of the dosing formulations. The test item dissolved in olive oil at concentration of 25 w/v% (non-GLP).
This vehicle is used for a general toxicity study and historical control data existed.
- Concentration in vehicle: 7.50, 2.50, 0.750 w/v%
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): 801029
Details on mating procedure:
- M/F ratio per cage: Each female was cohabited in the cage of a male in the same dose level, as a pair of male and female at night on the dosing day 15, and the females were returned to the original cage next morning. Cohabitation was continued until showing evidence of copulation.
- Length of cohabitation: maximum of 14 days.
- Proof of pregnancy: When a vaginal plug or sperms in vaginal smear were confirmed it was considered to be an evidence of mating (day 0 of pregnancy).
- After successful mating each pregnant female was caged (how): Copulated females were housed in polycarbonate cages (PC cage, 265 Wx426 Dx150 H mm, TOKIWA) with autoclaved wooden bedding, and enrichments of autoclaved gnawing wood and hemp mats from gestation day 14. Pups were housed with a dam after delivery.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of the test item formulations were confirmed with GC in the first preparation.
Actual concentrations of the 7.50, 2.50 and 0.750 w/v% formulations were 7.34, 2.42 and 0.717 w/v%, respectively.
Duration of treatment / exposure:
Male and female rats were treated with test item for 14 days and set on mating period for maximum of 14 days. Males and females were administrated until day 29 of dosing and 13 days after delivery, respectively.
Frequency of treatment:
Once daily. Three females were treated twice a day on day 39.
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Intermediate dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
10 male/ female/ dose
non-mating satellite group: 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A 14-day repeated-dose oral toxicity study was performed at the dose levels of 0, 50, 200, 500 and 1000 mg/kg/day consisting three males and three females for each group. Salivation was observed in the 200, 500 and 1000 mg/kg groups. Decreased spontaneous locomotion, decreased respiratory rate and incomplete eyelids opening were observed in the 500 and 1000 mg/kg groups. Staining lower abdomen, restlessness and staggering gait were observed in the 1000 mg/kg group. A decrease or a trend toward decrease in body weights were noted in the 500 and 1000 mg/kg groups. In organ weights, absolute and relative weights of the liver were increased and absolute weight of the spleen was decreased in the 500 and 1000 mg/kg groups. Absolute weights of the testes were decreased and relative weights of the kidneys were increased in the 1000 mg/kg group. In necropsy, enlargement of the liver was observed in the 500 and 1000 mg/kg groups. Apparent spotty pattern of the surface of the kidneys was observed in males of the 500 mg/kg group. Small testes and thymus, elevation of the limiting ridge of the forestomach were observed in the 1000 mg/kg group. Therefore 300 mg/kg/day was set for the high dose, and 100 and 30 mg/kg/day were set for the middle and low doses for the present study.
- Fasting period before blood sampling for clinical biochemistry: 16-20 h

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing period: Twice a day; Recovery period: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the dosing period and once a week thereafter.

SENSORIMOTOR FUNCTION EXAMINATIONS: yes
- Time schedule: The animals were examined for the parameters of the following listed in five males of each dose groups, non-mating five females of the control and 300 mg/kg groups, and selected five parental females of each dose group in each last dosing week. Not performed for the recovery period.
- Reflex (response to optic, pinna and pain, pupillary reflex, air righting reflex) tests and measurements of grip strengths were performed on a blind test basis. Locomotor activity of each animal was counted.


BODY WEIGHT: Yes
- Time schedule for examinations:
Males and non-mating females: Days 1, 3, 7, 14, 21 and 28 of dosing, days 1, 7 and 14 of recovery, and necropsy day.
Mating females: Days 1, 3, 7 and 14 of dosing, gestation days 0, 7, 14 and 20, postnatal days 0, 4, 8 and 13, and necropsy day.
Dead animals were weighed on the day found dead. Non-coupled female was weighed once a week after day 21 and necropsy day (day 55, equivalent pregnant day 26).


FOOD CONSUMPTION: yes
- Mean food consumption per day was calculated from their feeding and remainder weights.
- Time schedule for examinations:
Males and non-mating females: Days 1, 3, 7, 14, 21 and 28 of dosing, days 1, 7 and 14 of recovery
Mating females: Days 1, 3, 7 and 14 of dosing, gestation days 0, 7, 14 and 20, postnatal days 0, 4, 8 and 13


WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 16-20 h of the start of fasting
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: five males, five mating females for each dose group and non-mating five females and males and females of the recovery group
- Whole blood or plasma samples were examined. Parameters examined see "Examination parameters" in attached background material.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 16-20 h of the start of fasting
- Animals fasted: Yes
- How many animals: five males, five mating females for each dose group and non-mating five females and males and females of the recovery group
- Serum samples were used, Parameters examined see "Examination parameters" in attached background material.

URINALYSIS: Yes
- Time schedule for collection of urine: at afternoon of the each last observation day
- Metabolism cages used for collection of urine: Yes, Five males of the main group (same animals of sensorimotor function examinations), non-mating females, and male and females of the recovery group were housed in a metabolic cages at afternoon of the each last observation day with free drinking and no feed until next morning for 15-17 h.
- Animals fasted: Yes
- Parameters examined see "Examination parameters" in attached background material.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Vaginal smears of all females in the mating group were collected from day 1 to day 14 of dosing. Stages of the estrous cycle were determined with a light microscope after Giemsa staining.
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Clinical observations including stillborns were carried out once a day until postnatal day 13. The external surface including the palate of the live pups was observed on postnatal days0 and 13. Sex was determined for all pups on postnatal days 0, 4 and 13, and their body weight were measured on the same days. Anogenital distance (AGD) of each live pup was measured on postnatal day 4. Number of nipples/areolae of live male pups was counted on postnatal day 13.
Thyroid was collected from pups for T4 measurement on postnatal day 13 and fixed with 10% neutralized buffered formalin.

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):
SACRIFICE
- Survived parental animals were subjected to a detailed gross necropsy after bleeding from the ventral aorta under isoflurane anesthesia on the next day of last dosing and last recovery days.

GROSS NECROPSY
- Gross necropsy consisted of external surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues examined and weighed see "Examination parameters" in attached background material.
Statistics:
Data regarding body weights of parental animals, food consumption, grip strength, locomotor activity counts, parameters of the hematological and blood chemical examinations, urine volume, specific gravity of urine, organ weights, body weights on necropsy day, mean estrous cycle length, pairing days until copulation, gestation length, numbers of corpora lutea and implantation sites, numbers of pup born and live pup, body weights of pups, AGD and number of nipples/areolae were analyzed by the Barlett's test for homogeneity of variance. If significant difference (p<0.05) was not noted, analyzed by the Dunnett's test. If significant difference (p<0.05) was noted in the Bartlett test, analyzed by the nonparametric Dunnett's test. The frequencies of defecation and urination during the dosing period were analyzed by the nonparametric Dunnett's test.
Abnormal estrus cycle index, copulation index, conception index, delivery dam index and sex ratios were analyzed by the Fisher's exact test between the control group and each test item group.
Implantation index, delivery index, birth index and viability index were examined by the Bartlett's test for homogeneity of variance. If significant difference (p<0.05) was not noted, analyzed by the Dunnett's test. If significant difference (p<0.05) was noted in the Bartlett test, analyzed by the nonparametric Dunnett's test.
Data regarding body weights and food consumption during the recovery period, and parameters of the hematological and blood chemical examinations, urine volume, specific gravity of urine, organ weights and body weights on the necropsy day for the recovery group were analyzed by the F-test for variance ratio. If there were no significant differences at a significance level of 5%, the Student's t-test was performed. If there were significant differences, the Aspin-Welch t-test was performed. The frequencies of defecation and urination during the recovery period were analyzed by the Mann-Whitney U-test.
Reproductive indices:
- Copulation index: (number of copulated pairs / Number of mated pairs) x 100
- Conception index: (number of pregnant females / Number of copulated females) x 100
- Implantation index: (Number of implantation sites / Number of corpora lutea) × 100
-Delivery dam index: (Number of pregnant females with live pups / Number of pregnant females) × 100
- Delivery index: (Number of pups born / Number of implantation sites) x 100.
Offspring viability indices:
-Birth index: (Number of live pups I Number of implantation sites) × 100
- Viability index at birth: (Number of live pups at birth / Number of pups born) x 100
- Sex ratio of pups at birth: Number of live males at birth / Number of live pups at birth.
- Viability index on day 4 after birth: (Number of live pups on day 4 I Number of live pups at birth) x 100
- Sex ratio of pups on day 4 after birth: Number of males on day 4 I Number of live pups on day 4.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed immediately after dosing to males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group. Soft stool was observed in males and non-mating females of the 300 mg/kg group. Slight decreased spontaneous locomotion and lacrimation were observed in females of the 300 mg/kg group on day 22 of gestation period. During the recovery period soft stool was observed in males of the 300 mg/kg group and disappeared by day 3.
Detailed clinical observations were not affected by the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 300 mg/kg group, one animal was died during delivery (day 23 of the gestation period) and other animal was died on day 22 of the gestation period.
These animals did not show any severe changes compared to survived animals, and they were considered to have increased-stress related to delivery since small thymus and enlargement of the adrenals were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were decreased in males of the 100 and 300 mg/kg groups. During the recovery period body weights were decreased in males of the 300 mg/kg group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was decreased in males of the 100 and 300 mg/kg groups and females of the 300 mg/kg 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):
Hemoglobin conc. was decreased in males of the 300 mg/kg group. Prothrombin time was elongated in non-mating females of the 300 mg/kg group. At the end of the recovery period reticulocytes were increased in males of the 300 mg/kg group.
The test item affected the blood coagulation system.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, ALP was decreased in the 30, 100 and 300 mg/kg groups. ALT was decreased in the 100 and 300 mg/kg groups. T4 was increased in the 100 mg/kg group without dose-relationship. Potassium was decreased in the 300 mg/kg group.
In mating and non-mating females, ALT was decreased in the 300mg/kg group.
These changes were considered to be related to the decreases in body weights and food consumption, no toxicologically significant
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Renal tubular epithelial cells were observed in males of the 30, 100 and 300 mg/kg groups. Cloudy urine was observed in males of the 300 mg/kg group.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Grip strength of forelimbs was decreased to mating females of the 300 mg/kg group.
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):
Accumulation of the hyaline droplets in the proximal tubules, increased eosinophilic bodies and regeneration of the tubules in the outer medulla of the kidney were observed in males of the 30, 100 and 300 mg/kg groups.
Centrilobular hypertrophy of the hepatocytes of the liver was observed in males of the 100 and 300 mg/kg groups and females of the 300 mg/kg group. At the end of the recovery period granular cast and regeneration of the tubules in the outer medulla of the kidney were observed in males of the 300 mg/kg group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No abnormal estrus cycle was noted in any dose levels.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No abnormalities observed for estrus cycle stages at necropsy.
Gestation length was elongated in the 300 mg/kg group. A trend toward decrease in delivery dam index was noted in the 300 mg/kg group due to deaths of parental animals. These changes were considered to be related to aggravation of the general condition of dams.
Copulation index showed 90% of the control group in the 30 mg/kg group since copulation was not confirmed in one animal. This change was no dose dependent. Gestation length was also elongated in the 30 mg/kg group, and this change did not show a dose-relationship. Abnormal changes were not noted in the 100 mg/kg group.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other: for reproductive and developmental toxicity

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed

Details on results (F1)

Deaths were observed in the control, 30, 100 and 300 mg/kg groups, 15, 3, 5 and 8 pups, respectively. Trends toward decreases in birth index (91 % of the control group) viability index on postnatal day 0 (91 % of the control group), number of live pups (92% of the control group) and body weights (males: 91 % and females: 89% of corresponding control groups) on postnatal day 4, number of live pups on postnatal day 13 (89% of the control group) were noted in the 300 mg/kg group. No milk band and subnormal temperature were observed in one litter which all pups were died in the control group. Loss of tail tip was observed in one pup of the 300 mg/kg group. The test item did not affect any other developmental parameters of pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
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:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for the reproductive and developmental toxicity was considered to be 100 mg/kg/day due to elongation of gestation period, trends toward decreases in delivery dam index, birth index, viability index on postnatal day 0, number of live pups and body weights on postnatal day 4, number of live pups on postnatal day 13 in the 300 mg/kg group.
Executive summary:

"A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test" was performed to assess the repeated-dose, reproductive and developmental toxicities of test item in accordance with OECD 422.

Male and female Crl:CD(SD) rats at 9 weeks of age was treated with the test item for 14 days and set on mating period for maximum of 14 days. Males and females were administrated until day 29 of dosing and 13 days after delivery, respectively. The dosage levels were set at 0 (olive oil), 30, 100 and 300 mg/kg/day. Recovery groups were set for the 0 and 300 mg/kg groups after 29 days treatment for males and females of the satellite group.

In reproduction/developmental toxicity, gestation period was elongated in the 300 mg/kg group. A trend toward decrease in delivery dam index was noted in the 300 mg/kg group. Trends toward decreases in birth index, viability index on postnatal day 0, number of live pups and body weights on postnatal day 4, number of live pups on postnatal day 13 were noted in the 300 mg/kg group.

 

In conclusion, the No-Observed-Adverse-Effect Level (NOAEL) for repeated-dose toxicity under the conditions tested was considered to be 30 mg/kg/day due to decreases in body weights and food consumption, an increase in relative weight of the liver and centrilobular hypertrophy of the hepatocytes in the 100 and 300 mg/kg groups.

The NOAEL for the reproductive and developmental toxicity was considered to be 100 mg/kg/day due to elongation of gestation period, trends toward decreases in delivery dam index, birth index, viability index on postnatal day 0, number of live pups and body weights on postnatal day 4, number of live pups on postnatal day 13 in the 300 mg/kg group.