tert-butyl methacrylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 February 2007 to 12 September 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

7 February 2007 to 12 September 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

yes

Principles of method if other than guideline:

The pups were killed on day 4 after birth (no exposure until day 13 during the lactation period), no blood samples were taken for the T4 assessment, AGD and nipples reduction in pups were not examined, prostate and seminal vesicles with coagulating glands, uterus and cervix were not examined

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (supplier): MHLW Japan
- Batch number of test material: not specified
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool dark place (in a refrigerator, measured temperature range: 2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: It has been confirmed that 5 and 100 mg/ml test substance suspensions (vehicle: 0.1% Tween80-0.5% CMC-Na solution) that have been stored in a cool dark place (in a refrigerator, permitted range: 1-10°C) for 8 days, are then stable at room temperature for 24 hours.
- Solubility and stability of the test material in the solvent/vehicle: sparingly soluble in water, soluble in most organic solvents; stable under normal conditions with addition of polymerization; The results of an analysis of characteristics conducted by the test substance manufacturer on completion of a related study confirmed that there were no problems with the stability of the test substance

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 10, 30 and 100 mg/ml

FORM AS APPLIED IN THE TEST
- solution

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 318-364 g for males and 216-253 g for females
- Fasting period before study: not specified
- Housing: individually (or in pairs (1 male and 1 female; 2 animals in total) during the mating period)
- Diet: NMF (solid feed, sterilized by irradiation), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22-26°C
- Humidity: 37-55%
- Air changes: 10-15 times per hour
- Photoperiod: illumination 12 hours a day (07:00-19:00)

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The specified concentrations (10, 30 and 100 mg/ml) were obtained by weighing out the amount of test substance required for the respective concentration, then suspending it in the vehicle, using a homogenizer, until emulsified, and then making the emulsion up to the specified volume. The volume administered was set at 10 ml/kg body weight.

- Rate of preparation (frequency): Test solution preparation was performed 9 times in total (on February 21, 27, March 6, 13, 20, 27, 29, and April 6 and 13, 2007)
- Storage conditions: The solutions to be administered (including the control group solution) were dispensed into brown glass bottles (light-shielding bottles) in daily dose aliquots, stored in a cool dark place (in a refrigerator, measured values: 2-6°C), and administered within 8 days of preparation.

VEHICLE
- Vehicle constituent 1: Carmellose sodium (CMC-Na)
- Lot No.: 6220
- Purity: not specified

- Vehicle constituent 2: Water for injection
- Lot No.: 6K76, 6K90
- Purity: not specified

- Vehicle constituent 3: Tween®80
- Lot No.: 7398H
- Purity: not specified

First, CMC-Na was dissolved in water for injection to obtain 0.5 w/v%, then Tween®80 was added to obtain 0.1 v/v%, and then the solution was made up to the specified volume, to obtain 0.1% Tween80-0.5% CMC-Na solution. The control substance (vehicle) was prepared the day before preparation of the respective test substance solution, and was introduced into a glass container and stored in a cool place (in a refrigerator, measured values: 1-9°C) until use.

- Justification for use and choice of vehicle(s): not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test solutions of each concentration that was administered to males in week 1 and in the final week of administration were analyzed by the GC method at BoZo Research Center. The results revealed that these concentrations were 93.0-98.3% of the nominal value, and the C.V. was 0.2- 0.7%, thus both were within the permitted range (concentration: 100±10% of nominal value, C.V.: ≤10%).

Details on mating procedure:

- M/F ratio per cage: On completion of the pre-mating administration period, males and females from the same administration group were housed 1:1 in the same cage overnight.
- Proof of pregnancy: Vaginal plug formation or the detection of sperm in the vaginal smear the following morning was regarded as confirmation that copulation had occurred. The number of days until copulation was counted, taking the day the mating period started as day 0.
- After successful mating each pregnant female was caged (how): individually

Duration of treatment / exposure:

The administration period for males was the 42-day period comprising the 14-day pre-mating period, the 14-day mating period, plus 14 days after completion of the mating period. The administration period for females was the 42- to 48-day period comprising the 14-day pre-mating period, the mating period, plus the gestation period through to day 4 of nursing. For one female that did not copulate the administration period was 53 days.

Frequency of treatment:

daily, 7 days each week (at 8:42-12:14, although for animals that were giving birth at the administration time, administration was performed after delivery, at 17:00-17:23)

Duration of test:

similar to duration of treatment

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

Dose / conc.:

300 mg/kg bw/day

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

Dose / conc.:

100 mg/kg bw/day

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary study conducted previously, no animal death was observed, but 1000 mg/kg (the administration limit in OECD Guideline for Testing of Chemicals 421)test substance administration did have a mild effect on body weight, feed intake and livers. No effects of test substance administration were observed in the ≥ 300 mg/kg dose groups. Accordingly, three doses were set for this simple reproductive toxicity study: 1000 mg/kg as the high dose, thereafter dividing by a common ratio of 3, to give 300 and 100 mg/kg.
- Rationale for animal assignment: For group allocation, the animals were stratified according to body weight on the day of allocation (the day before administration), and assigned to groups so that the mean body weight in each group was as uniform as possible. The allocation was performed by computer using a combination of the block placement method and the random extraction method (required groups were assembled by the block placement method, and test groups and individual numbers within the group were randomly assigned).

Maternal examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Three times a day (before administration, immediately after administration and about 2 hours after administration) throughout the administration period, the general condition of all animals was observed for any abnormality in external body surface, nutritional state, posture, behavior, excrement, etc.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights were measured on administration days 1, 4, 8, 11, 15 (also on administration days 18, 22, 25, 29, 33, 36, 40, 43, 46 and 49 for animals that did not copulate) and pregnancy days 0, 4, 7, 11, 14, 17 and 20 and nursing days 0and 4. The body weight was measured at 8:32-11:27, except for the nursing day 0 body weight if delivery completion was confirmed in the afternoon delivery observation (in which case the body
weight was measured between 14:37 and 17:11).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Remaining feed quantity was measured for all animals: on administration days 1, 4, 8, 11, 15 (and also on administration days 30, 33, 36, 40, 43, 46 and 49 for animals that did not copulate), and on pregnancy days 1, 4, 7, 11, 14, 17 and 20 and nursing days 2 and 4. The daily feed intake per animal was calculated from the feed intake the previous day. The quantity of feed and the amount of feed remaining were measured between 08:32 and 11:31.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 5
- Organs examined: ovaries, uterus, vagina, visibly abnormal sites (liver and kidneys), individual identification site (auricle)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Blood sampling:

not examined

Fetal examinations:

The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths/ live births/ postnatal mortality
- presence of gross anomalies,
- body weight of each individual liveborn pup was determined, and the mean values for males and females in each litter were calculated

Statistics:

The body weights, feed intakes, numbers of estrous images, estrous cycles, gestation periods, numbers of corpora lutea, numbers of implantation marks, numbers of surviving pups and organ weights were tested by the following methods: mean value for each group ± SD, Bartlett test/F test and Dunnett’s test/Dunnett-type test/Student t-test/Aspin-Welch t-test.
The implantation index, stillbirth index, livebirth index, external abnormality index and viability index of liveborn pups were determined for each animal, and then homogeneity of variance was tested using the Bartlett test (significance level 0.01, both sides). If homogeneous, Dunnett’s test was performed, and if non homogeneous, a Dunnett-type test was performed (significance level 0.05 and 0.01, both sides). To determine the conception index and delivery index, and the sex ratio of the liveborn pups, for each group the total number of animals that copulated, number of males that impregnated a female, number of pregnant females, number of females that delivered live pups, number of surviving male pups, and number of surviving female pups were determined, and the x2 test with Yates’ continuity correction was conducted (significance level 0.05 and 0.01, both sides). When a cell with expected frequency of ≤5 was observed, the test was conducted using Fisher’s direct probability method (significance level 0.05 and 0.01, both sides).

Indices:

Stillbirth index (%) = (number of stillborn pups/total number of pups born) x 100;
Livebirth index (%) = (number of liveborn pups/total number of pups born) x 100;
External abnormality index (%) = (number of pups with external abnormality/number of liveborn pups) x 100;
Sex ratio = number of male pups/(number of male pups + number of female pups);
Viability index of liveborn pups (%) = (number of surviving pups on nursing day 4/number of liveborn pups on nursing day 0) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no deaths in any administration group, and no abnormalities in general condition were observed.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the administration period the body weights in all administration groups were equivalent to those in the control group, and no significant difference was found.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious difference in the feed intake of the females in the 100 mg/kg group. Females in the 300 mg/kg group had a significantly high feed intake on pregnancy day 11. In the 1000 mg/kg group, the females had a significantly low feed intake on administration day 4 and a significantly high feed intake on pregnancy day 11. However, there was no abnormal body weight variation, and so these changes were deemed to have no toxicological significance.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes attributed to test substance administration were seen in the livers and kidneys. Kidney: Hypertrophy was observed 1 female in the 300 mg/kg group and 7 females in the 1000 mg/kg group. Liver: Hypertrophy was observed in 2 females in the 100 mg/kg group, 7 females in the 300 mg/kg group and 11 females in the 1000 mg/kg group. Findings were observed in the following organs and tissues, however, based on the appearance frequency and pathological characteristics, these were thought to be accidental changes. Kidney: Depressed focus was observed in 1 female in the 1000 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes attributed to test substance administration were seen in the livers and kidneys.
- Liver: Minimal brightening of hepatocytes was observed in 2/2 females in the 100 mg/kg group, 4/7 females in the 300 mg/kg group and 8/11 females in the 1000 mg/kg group. Minimal or mild centrilobular hepatocytes hypertrophy was observed in 2/7 females in the 300 mg/kg group and 8/11 females in the 1000 mg/kg group. Minimal localized necrosis was observed in 3/11 females in the 1000 mg/kg group. - Kidney: Minimal or mild renal tubule dilatation was observed in 5/8 females in the 1000 mg/kg group. Minimal or mild renal tubule vacuolation was observed in 5/8 females in the 1000 mg/kg group. Minimal or mild renal tubule necrosis was observed in 3/8 females in the 1000 mg/kg group. Minimal or mild hyaline cast was observed in 1/8 females in the 1000 mg/kg group. Minimal or mild renal tubular basophilia was observed in 4/8 females in the 1000 mg/kg group. Minimal or mild glomerular hyaline droplets was observed in 6/8 females in the 1000 mg/kg group. Based on the onset circumstances and histologic characteristics, the following findings in various organs and tissues were considered to be accidental changes.
- Liver: Minimal microgranuloma was observed in 1 female in the 1000 mg/kg group.
- Kidney: Minimal localized fibrosis was observed in 1 female in the 1000 mg/kg group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Number of abortions:

not examined

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Normal delivery was achieved on pregnancy day 21.5-22.5, in all cases.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

No significant differences were observed between the control group and any administration group .

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

< 100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Changes attributed to test substance administration were seen in the livers and kidneys.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no significant differences between the control group and any administration group in the number of liveborn pups.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no significant differences between the control group and any administration group in the sex ratio.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

The birth weight was significantly high in females in the 100 mg/kg group, but this was not dose-related, and the change was not toxicologically significant. There were no other significant differences between the control group and any administration group.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

The only pup deaths during the nursing period were 7 pup deaths in the control group, 8 in the 300 mg/kg group and 4 in the 1000 mg/kg group, and there was no significant difference between the control group and any administration group in the viability index on nursing day 4.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

As external abnormality, 1 case of omphalocele and 1 case of crooked tail were observed in the 1000 mg/kg group; both changes were observed at low frequency and both were thought to be accidental changes.

Skeletal malformations:

not examined

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Thymic remnant in the neck were observed in 1 male in the control group, 1 male in the 100 mg/kg group, 1 male in the 300 mg/kg group, and 1 male and 3 females in the 1000 mg/kg group; this change occurred at low frequency, and, based on the developmental characteristics, was thought to be an accidental change. In addition, situs inversus totalis was observed in 1 female in the control group.

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

for developmental toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance was less than 100 mg/kg/day in females, and that the no-observed effect dose with respect to reproduction and development toxicity was 1000 mg/kg/day in the female parents and pups.

Executive summary:

The test substance was administered at 0 (control group: aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added), 100, 300 and 1000 mg/kg to Sprague- Dawley SPF rats, for 14 days before mating and throughout the mating period until the day before autopsy in males (42 days), and for 14 days before mating and throughout the mating period and gestation period until day 4 of nursing in females (42-48 days), and the repeated dose toxicity and reproductive and developmental toxicity were investigated according to a OECD TG 421 study. In the parent animals, test substance administration had no observable effect on estrous cycle, days required until copulation, copulation index, insemination index or conception index. Furthermore, test substance administration had no observable effect on delivery index, gestation period, number of corpora lutea, number of implantation marks, implantation index, stillbirth index, number of liveborn pups, livebirth index or sex ratio, and no abnormalities in nursing condition were observed. Therefore, test substance administration was thought to have had no effect on reproductive functions such as copulation ability in males or females, or on insemination ability or conception ability, or on pregnancy maintenance, delivery and nursing behavior, etc. of dams, even in the 1000 mg/kg group. In the liveborn pups, no changes due to test substance administration were observed in body weight at birth or on day 4 of nursing, in males or females, and no changes due to test substance administration were observed on external examination at birth, or in the autopsy findings on day 4 of nursing, or in the viability index. It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance in females was less than 100 mg/kg/day, and that the no-observed effect dose with respect to reproduction and development toxicity in female parents and pups was 1000 mg/kg/day.

Cross-reference 2

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:

7 February 2007 to 12 September 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

yes

Principles of method if other than guideline:

The pups were killed on day 4 after birth (no exposure until day 13 during the lactation period), no blood samples were taken for the T4 assessment, AGD and nipples reduction in pups were not examined, prostate and seminal vesicles with coagulating glands, uterus and cervix were not examined

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (supplier): MHLW Japan
- Batch number of test material: not specified
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool dark place (in a refrigerator, measured temperature range: 2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: It has been confirmed that 5 and 100 mg/ml test substance suspensions (vehicle: 0.1% Tween80-0.5% CMC-Na solution) that have been stored in a cool dark place (in a refrigerator, permitted range: 1-10°C) for 8 days, are then stable at room temperature for 24 hours.
- Solubility and stability of the test material in the solvent/vehicle: sparingly soluble in water, soluble in most organic solvents; stable under normal conditions with addition of polymerization; The results of an analysis of characteristics conducted by the test substance manufacturer on completion of a related study confirmed that there were no problems with the stability of the test substance

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 10, 30 and 100 mg/ml

FORM AS APPLIED IN THE TEST
- solution (test substance was suspended in 0.5% aqueous CMC-Na solution with 0.1% Tween 80-added)

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Details on species / strain selection:

Rat was selected as the animal species, in accordance with toxicity study guidelines. The strain of rat used in the present study was chosen because it is widely used in general toxicity studies and reproductive and developmental toxicity studies, the characteristics of the strain are well known, and there is a lot of background data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 318-364 g for males and 216-253 g for females
- Fasting period before study: not specified
- Housing: individually (or in pairs (1 male and 1 female; 2 animals in total) during the mating period)
- Diet: NMF (solid feed, sterilized by irradiation), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY:
- As far as contaminants in the feed were concerned, the results of analyses conducted by Japan Food Research Laboratories were obtained for the lots used. For the drinking water, analysis of water quality in accordance with the Water Supply Act was requested periodically (4 times a year) from Toshiba Machine Environment Center, and the results were obtained. It was confirmed from these analysis results that there was no possibility of contaminants in the feed and drinking water affecting the study results.

ENVIRONMENTAL CONDITIONS
- Temperature: 22-26°C
- Humidity: 37-55%
- Air changes: 10-15 times per hour
- Photoperiod: illumination 12 hours a day (07:00-19:00)

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Details on route of administration:

Oral administration was selected as the administration route, in accordance with OECD Guideline for Testing of Chemicals 421.

Vehicle:

other: Aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The specified concentrations (10, 30 and 100 mg/ml) were obtained by weighing out the amount of test substance required for the respective concentration, then suspending it in the vehicle, using a homogenizer, until emulsified, and then making the emulsion up to the specified volume. The volume administered was set at 10 ml/kg body weight.

- Rate of preparation (frequency): Test solution preparation was performed 9 times in total (on February 21, 27, March 6, 13, 20, 27, 29, and April 6 and 13, 2007)
- Storage conditions: The solutions to be administered (including the control group solution) were dispensed into brown glass bottles (light-shielding bottles) in daily dose aliquots, stored in a cool dark place (in a refrigerator, measured values: 2-6°C), and administered within 8 days of preparation.

VEHICLE
- Vehicle constituent 1: Carmellose sodium (CMC-Na)
- Lot No.: 6220
- Purity: not specified

- Vehicle constituent 2: Water for injection
- Lot No.: 6K76, 6K90
- Purity: not specified

- Vehicle constituent 3: Tween®80
- Lot No.: 7398H
- Purity: not specified

First, CMC-Na was dissolved in water for injection to obtain 0.5 w/v%, then Tween®80 was added to obtain 0.1 v/v%, and then the solution was made up to the specified volume, to obtain 0.1% Tween80-0.5% CMC-Na solution. The control substance (vehicle) was prepared the day before preparation of the respective test substance solution, and was introduced into a glass container and stored in a cool place (in a refrigerator, measured values: 1-9°C) until use.

- Justification for use and choice of vehicle(s): not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test solutions of each concentration that was administered to males in week 1 and in the final week of administration were analyzed by the GC method at BoZo Research Center. The results revealed that these concentrations were 93.0-98.3% of the nominal value, and the C.V. was 0.2- 0.7%, thus both were within the permitted range (concentration: 100±10% of nominal value, C.V.: ≤10%).

Duration of treatment / exposure:

The administration period for males was the 42-day period comprising the 14-day pre-mating period, the 14-day mating period, plus 14 days after completion of the mating period. The administration period for females was the 42- to 48-day period comprising the 14-day pre-mating period, the mating period, plus the gestation period through to day 4 of nursing. For one female that did not copulate the administration period was 53 days.

Frequency of treatment:

daily, 7 days each week (at 8:42-12:14, although for animals that were giving birth at the administration time, administration was performed after delivery, at 17:00-17:23)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

Dose / conc.:

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

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

Dose / conc.:

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

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary study conducted previously, no animal death was observed, but 1000 mg/kg (the administration limit in OECD Guideline for Testing of Chemicals 421)test substance administration did have a mild effect on body weight, feed intake and livers. No effects of test substance administration were observed in the ≥ 300 mg/kg dose groups. Accordingly, three doses were set for this simple reproductive toxicity study: 1000 mg/kg as the high dose, thereafter dividing by a common ratio of 3, to give 300 and 100 mg/kg.
- Rationale for animal assignment: For group allocation, the animals were stratified according to body weight on the day of allocation (the day before administration), and assigned to groups so that the mean body weight in each group was as uniform as possible. The allocation was performed by computer using a combination of the block placement method and the random extraction method (required groups were assembled by the block placement method, and test groups and individual numbers within the group were randomly assigned).
- Rationale for selecting satellite groups: no satellite groups available
- Post-exposure recovery period in satellite groups: no satellite groups available

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Three times a day (before administration, immediately after administration and about 2 hours after administration) throughout the administration period, the general condition of all animals was observed for any abnormality in external body surface, nutritional state, posture, behavior, excrement, etc.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the males were measured on administration days 1, 4, 8, 11, 15, 18, 22, 25, 29,32, 36, 39 and 42 and the autopsy day, and the body weights of the females were measured on administration days 1, 4, 8, 11, 15 (also on administration days 18, 22, 25, 29, 33, 36, 40, 43, 46 and 49 for animals that did not copulate) and pregnancy days 0, 4, 7, 11, 14, 17 and 20 and nursing days 0and 4. The body weight was measured at 8:32-11:27, except for the nursing day 0 body weight if delivery completion was confirmed in the afternoon delivery observation (in which case the body
weight was measured between 14:37 and 17:11).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Remaining feed quantity was measured for all animals: for males on administration days 1, 4, 8, 11, 15, 32, 36, 39 and 42; and for females on administration days 1, 4, 8, 11, 15 (and also on administration days 30, 33, 36, 40, 43, 46 and 49 for animals that did not copulate), and on pregnancy days 1, 4, 7, 11, 14, 17 and 20 and nursing days 2 and 4. The daily feed intake per animal was calculated from the feed intake the previous day. The quantity of feed and the amount of feed remaining were measured between 08:32 and 11:31.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY: not examined

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE
- All animals: on the day after the final administration

GROSS NECROPSY AND ORGAN WEIGHTS
Animals were euthanized by exsanguination via abdominal aortic section under ether anesthesia and then detailed pathological autopsies comprising visual examination of systemic organs and tissues including the external body surface, head, chest and abdomen were conducted, and the results were recorded. As abnormalities were observed in the liver and kidneys in the test substance administration groups, 3 animals from the control group were stored in the same way. Also, the number of corpora lutea and implantation marks on nursing day 5 were counted for the females (dams). For the males, the weights (absolute weights) of the testes and epididymis were measured, and the relative weights per 100 g body weight were calculated from the absolute weight and body weight at the time of autopsy. For bilateral organs, measurements were taken for the left and the right organ, and evaluation based on the total thereof.

HISTOPATHOLOGY
For all animals, the following organs and tissues were fixed using phosphate buffered 10 vol% formalin solution and stored (testes and epididymis were fixed using Bouin’s solution and then stored in phosphate buffered 10 vol% formalin solution). Next, samples were embedded in paraffin, and then sectioned, and stained using hematoxylin-eosin (HE). Of the resulting samples, the testes, epididymis, ovaries, and any visibly abnormal sites of the control group and high dose group animals were examined under microscope (for bilateral organs, both were extracted, and one of them was examined). Effects of test substance administration were observed in the livers and kidneys of males and females, which were visibly abnormal (with reference to the same sites of control group animals), and so typical examples of normal and abnormal findings were photographed.
-Testes, epididymis, prostate gland, seminal vesicle, ovaries, uterus, vagina, visibly abnormal sites (liver and kidneys), individual identification site (auricle)

Statistics:

The body weights, feed intakes and organ weights were tested by the following methods: mean value for each group ± SD, Bartlett test/F test and Dunnett’s test/Dunnett-type test/Student t-test/Aspin-Welch t-test.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities in general condition were observed.

Mortality:

no mortality observed

Description (incidence):

There were no deaths in any administration group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the administration period the body weights of males and females in all administration groups were equivalent to those in the control group, and no significant difference was found.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious difference in the feed intake of the males or females in the 100 mg/kg group. Females in the 300 mg/kg group had a significantly high feed intake on pregnancy day 11. In the 1000 mg/kg group, the males had a significantly high feed intake on administration day 42 and in the females had a significantly low feed intake on administration day 4 and a significantly high feed intake on pregnancy day 11. However, there was no abnormal body weight variation, and so these changes were deemed to have no toxicological significance.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and relative weights of the testes and epididymis exhibited no obvious changes in any administration group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Changes attributed to test substance administration were seen in the livers and kidneys of males and females. Kidney: Hypertrophy was observed in 1 male in the 100 mg/kg group, 6 males and 1 female in the 300 mg/kg group, and 12 males and 7 females in the 1000 mg/kg group. Liver: Hypertrophy was observed in 5 males and 2 females in the 100 mg/kg group, 10 males and 7 females in the 300 mg/kg group, and 12 males and 11 females in the 1000 mg/kg group. Findings were observed in the following organs and tissues, however, based on the appearance frequency and pathological characteristics, these were thought to be accidental changes. Epididymis: White focus was observed in 1 animal in the 300 mg/kg group and in 1 animal in the 1000 mg/kg group. Kidney: Depressed focus was observed in 1 female in the 1000 mg/kg group. Liver: Diaphragmatic hernia nodule was observed in 1 male in the control group.Testis: Atrophy was observed in 1 animal in the 300 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes attributed to test substance administration were seen in the livers and kidneys.
- Liver: Minimal brightening of hepatocytes was observed in 2/2 females in the 100 mg/kg group, 4/7 females in the 300 mg/kg group and 8/11 females in the 1000 mg/kg group. Minimal or mild centrilobular hepatocytes hypertrophy was observed in 5/5 males in the 100 mg/kg group, 10/10 males and 2/7 females in the 300 mg/kg group, and 12/12 males and 8/11 females in the 1000 mg/kg group. Minimal localized necrosis was observed in 3/11 females in the 1000 mg/kg group. - Kidney: Minimal or mild renal tubule dilatation was observed in 1/1 male in the 100 mg/kg group, 3/6 males in the 300 mg/kg group, and 12/12 males and 5/8 females in the 1000 mg/kg group. Minimal or mild renal tubule vacuolation was observed in 5/8 females in the 1000 mg/kg group. Minimal or mild renal tubule necrosis was observed in 3/8 females in the 1000 mg/kg group. Minimal or mild hyaline cast was observed in 2/6 males in the 300 mg/kg group, and 10/12 males and 1/8 females in the 1000 mg/kg group. Minimal erythrocyte cast was observed in 1/1 male in the 100 mg/kg group, 3/6 males in the 300 mg/kg group, and 6/12 males in the 1000 mg/kg group. Minimal or mild renal tubular basophilia was observed in 1/1 male in the 100 mg/kg group, 2/6 males in the 300 mg/kg group, and 12/12 males and 4/8 females in the 1000 mg/kg group. Minimal or mild renal tubule hyaline droplets was observed in 1/1 male in the 100 mg/kg group, 6/6 males in the 300 mg/kg group, and 6/12 males in the 1000 mg/kg group. Minimal or mild glomerular hyaline droplets was observed in 1/1 male in the 100 mg/kg group, 3/6 males in the 300 mg/kg group, and 12/12 males and 6/8 females in the 1000 mg/kg group. Based on the onset circumstances and histologic characteristics, the following findings in various organs and tissues were considered to be accidental changes.
- Epididymis: Mild spermatic granuloma was observed in 1 animal in the 300 mg/kg group and in 1 animal in the 1000 mg/kg group.
- Ovary: Mild follicular cyst was observed in 1 animal in the 1000 mg/kg group.
- Testis: Mild seminiferous tubular atrophy was observed in 1 animal in the control group, and moderate seminiferous tubular atrophy was observed in 1 animal in the 300 mg/kg group.
- Liver: Mild diaphragmatic hernia nodule was observed in 1 male in the control group, and minimal microgranuloma was observed in 1 female in the 1000 mg/kg group.
- Kidney: Minimal localized fibrosis was observed in 1 female in the 1000 mg/kg group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

<= 100 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

no

Conclusions:

It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance was less than 100 mg/kg/day in males and females.

Executive summary:

The test substance was administered at 0 (control group: aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added), 100, 300 and 1000 mg/kg to Sprague-Dawley SPF rats, for 14 days before mating and throughout the mating period until the day before autopsy in males (42 days), and for 14 days before mating and throughout the mating period and gestation period until day 4 of nursing in females (42-48 days), and the repeated dose toxicity and reproductive and developmental toxicity were investigated according to a OECD TG 421 study. No animal deaths or abnormality in general condition were observed in any administration group. No effects of test substance administration on body weight or feed intake were observed in any administration group.On pathological examination, effects of test substance administration were found in the livers and
kidneys of males and females in the ≥100 mg/kg groups. In the livers there was visible hypertrophy, and histologically, centrilobular hepatocyte hypertrophy, brightening of hepatocytes and localized necrosis of hepatocytes were observed. The centrilobular hepatocyte hypertrophy was thought to be an adaptive change accompanying extracorporeal excretion of the present agent; the brightening of hepatocytes was attributed to glycogen accumulation under satiation; and the necrosis of hepatocytes was a localized lesion in each case. The histological characteristics and degrees of these changes were similar to those of naturally occurring changes in rats of the same strain, and no organic disorders were observed males or females, and so these changes were thought to have no toxicological significance. In the kidneys there was visible hypertrophy, and histologically, glomerular hyaline droplets, renal tubule hyaline droplets, basophilic change, dilatation, necrosis and vacuolation were observed; hyaline cast and erythrocyte cast were also observed. All renal tubule necrosis was accompanied by vacuolation. It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance in males and females was less than 100 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Principles of method if other than guideline:

The pups were killed on day 4 after birth (no exposure until day 13 during the lactation period), no blood samples were taken for the T4 assessment, AGD and nipples reduction in pups were not examined, prostate and seminal vesicles with coagulating glands, uterus and cervix were not examined

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl methacrylate

EC Number:

209-548-7

EC Name:

tert-butyl methacrylate

Cas Number:

585-07-9

Molecular formula:

C8H14O2

IUPAC Name:

tert-butyl 2-methylprop-2-enoate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (supplier): MHLW Japan
- Batch number of test material: not specified
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool dark place (in a refrigerator, measured temperature range: 2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: It has been confirmed that 5 and 100 mg/mL test substance suspensions (vehicle: 0.1% Tween80-0.5% CMC-Na solution) that have been stored in a cool dark place (in a refrigerator, permitted range: 1-10°C) for 8 days, are then stable at room temperature for 24 hours.
- Solubility and stability of the test material in the solvent/vehicle: sparingly soluble in water, soluble in most organic solvents; stable under normal conditions with addition of polymerization; The results of an analysis of characteristics conducted by the test substance manufacturer on completion of a related study confirmed that there were no problems with the stability of the test substance

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 10, 30 and 100 mg/ml

FORM AS APPLIED IN THE TEST
- solution

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Details on species / strain selection:

Rat was selected as the animal species, in accordance with toxicity study guidelines. The strain of rat used in the present study was chosen because it is widely used in general toxicity studies and reproductive and developmental toxicity studies, the characteristics of the strain are well known, and there is a lot of background data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks
- Weight at study initiation: 318-364 g for males and 216-253 g for females
- Fasting period before study: not specified
- Housing: individually (or in pairs (1 male and 1 female; 2 animals in total) during the mating period)
- Diet: NMF (solid feed, sterilized by irradiation), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22-26°C
- Humidity: 37-55%
- Air changes: 10-15 times per hour
- Photoperiod: illumination 12 hours a day (07:00-19:00)

IN-LIFE DATES: not specified

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The specified concentrations (10, 30 and 100 mg/mL) were obtained by weighing out the amount of test substance required for the respective concentration, then suspending it in the vehicle, using a homogenizer, until emulsified, and then making the emulsion up to the specified volume. The volume administered was set at 10 mL/kg body weight.

- Rate of preparation (frequency): Test solution preparation was
performed 9 times in total (on February 21, 27, March 6, 13, 20, 27, 29, and April 6 and 13, 2007)
- Storage conditions: The solutions to be administered (including the control group solution) were dispensed into brown glass bottles (light-shielding bottles) in daily dose aliquots, stored in a cool dark place (in a refrigerator, measured values: 2-6°C), and administered within 8 days of preparation.

VEHICLE
- Vehicle constituent 1: Carmellose sodium (CMC-Na)
- Lot No.: 6220
- Purity: not specified

- Vehicle constituent 2: Water for injection
- Lot No.: 6K76, 6K90
- Purity: not specified

- Vehicle constituent 3: Tween®80
- Lot No.: 7398H
- Purity: not specified

First, CMC-Na was dissolved in water for injection to obtain 0.5 w/v%, then Tween®80 was added to obtain 0.1 v/v%, and then the solution was made up to the specified volume, to obtain 0.1% Tween80-0.5% CMC-Na solution. The control substance (vehicle) was prepared the day before preparation of the respective test substance solution, and was introduced into a glass container and stored in a cool place (in a refrigerator, measured values: 1-9°C) until use.

- Justification for use and choice of vehicle(s): not specified

Details on mating procedure:

- M/F ratio per cage: On completion of the pre-mating administration period, males and females from the same administration group were housed 1:1 in the same cage overnight.
- Proof of pregnancy: Vaginal plug formation or the detection of sperm in the vaginal smear the following morning was regarded as confirmation that copulation had occurred. The number of days until copulation was counted, taking the day the mating period started as day 0.
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test solutions of each concentration that was administered to males in week 1 and in the final week of administration were analyzed by the GC method at BoZo Research Center. The results revealed that these concentrations were 93.0-98.3% of the nominal value, and the C.V. was 0.2- 0.7%, thus both were within the permitted range (concentration: 100±10% of nominal value, C.V.: ≤10%).

Duration of treatment / exposure:

The administration period for males was the 42-day period comprising the 14-day pre-mating period, the 14-day mating period, plus 14 days after completion of the mating period. The administration period for females was the 42- to 48-day period comprising the 14-day pre-mating period, the mating period, plus the gestation period through to day 4 of nursing. For one female that did not copulate the administration period was 53 days.

Frequency of treatment:

daily, 7 days each week (at 8:42-12:14, although for animals that were giving birth at the administration time, administration was performed after delivery, at 17:00-17:23)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the body weight on day 7 of pregnancy.

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Remarks:

The volume administered to each animal was calculated based on the most recent
body weight. For females from day 7 of pregnancy onwards, the volume was calculated based on the
body weight on day 7 of pregnancy.

No. of animals per sex per dose:

12/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary study conducted previously, no animal death was observed, but 1000 mg/kg (the administration limit in OECD Guideline for Testing of Chemicals 421) test substance administration did have a mild effect on body weight, feed intake and livers. No effects of test substance administration were observed in the ≥ 300 mg/kg dose groups. Accordingly, three doses were set for this simple reproductive toxicity study: 1000 mg/kg as the high dose, thereafter dividing by a common ratio of 3, to give 300 and 100 mg/kg.
- Rationale for animal assignment: For group allocation, the animals were stratified according to body weight on the day of allocation (the day before administration), and assigned to groups so that the mean body weight in each group was as uniform as possible. The allocation was performed by computer using a combination of the block placement method and the random extraction method (required groups were assembled by the block placement method, and test groups and individual numbers within the group were randomly assigned).

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Three times a day (before administration, immediately after administration and about 2 hours after administration) throughout the administration period, the general condition of all animals was observed for any abnormality in external body surface, nutritional state, posture, behavior, excrement, etc.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the males were measured on administration days 1, 4, 8, 11, 15, 18, 22, 25, 29,32, 36, 39 and 42 and the autopsy day, and the body weights of the females were measured on administration days 1, 4, 8, 11, 15 (also on administration days 18, 22, 25, 29, 33, 36, 40, 43, 46 and 49 for animals that did not copulate) and pregnancy days 0, 4, 7, 11, 14, 17 and 20 and nursing days 0and 4. The body weight was measured at 8:32-11:27, except for the nursing day 0 body weight if delivery completion was confirmed in the afternoon delivery observation (in which case the body
weight was measured between 14:37 and 17:11).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Remaining feed quantity was measured for all animals: for males on administration days 1, 4, 8, 11, 15, 32, 36, 39 and 42; and for females on administration days 1, 4, 8, 11, 15 (and also on administration days 30, 33, 36, 40, 43, 46 and 49 for animals that did not copulate), and on pregnancy days 1, 4, 7, 11, 14, 17 and 20 and nursing days 2 and 4. The daily feed intake per animal was calculated from the feed intake the previous day. The quantity of feed and the amount of feed remaining were measured between 08:32 and 11:31.

WATER CONSUMPTION AND COMPOUND INTAKE: No

Oestrous cyclicity (parental animals):

A vaginal smear sample was obtained for all females, every day from the day after the start of administration until confirmation of copulation; the samples were examined under a microscope. During the premating administration period, the smear images were classified into pro-estrous, estrous, metestrous and anestrous phases, and the number of estrous images and the number of days from one estrous phase until the next estrous phase (the estrous cycle) were investigated. During the mating period, the presence or absence of sperm in the smear was examined.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis and epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths/ live births/ postnatal mortality
- presence of gross anomalies,
- body weight of each individual liveborn pup was determined, and the mean values for males and females in each litter were calculated

Postmortem examinations (parental animals):

SACRIFICE
- All male and maternal animals: on the day after the final administration

GROSS NECROPSY AND ORGAN WEIGHTS
Animals were euthanized by exsanguination via abdominal aortic section under ether anesthesia and then detailed pathological autopsies comprising visual examination of systemic organs and tissues including the external body surface, head, chest and abdomen were conducted, and the results were recorded. As abnormalities were observed in the liver and kidneys in the test substance administration groups, 3 animals from the control group were stored in the same way. Also, the number of corpora lutea and implantation marks on nursing day 5 were counted for the females (dams). For the males, the weights (absolute weights) of the testes and epididymis were measured, and the relative weights per 100 g body weight were calculated from the absolute weight and body weight at the time of autopsy. For bilateral organs, measurements were taken for the left and the right organ, and evaluation based on the total thereof.

HISTOPATHOLOGY
For all animals, the following organs and tissues were fixed using phosphate buffered 10 vol% formalin solution and stored (testes and epididymis were fixed using Bouin’s solution and then stored in phosphate buffered 10 vol% formalin solution). Next, samples were embedded in paraffin, and then sectioned, and stained using hematoxylin-eosin (HE). Of the resulting samples, the testes, epididymis, ovaries, and any visibly abnormal sites of the control group and high dose group animals were examined under microscope (for bilateral organs, both were extracted, and one of them was examined). Effects of test substance administration were observed in the livers and kidneys of males and females, which were visibly abnormal (with reference to the same sites of control group animals), and so typical examples of normal and abnormal findings were photographed.
-Testes, epididymis, prostate gland, seminal vesicle, ovaries, uterus, vagina, visibly abnormal sites (liver and kidneys), individual identification site (auricle)

Postmortem examinations (offspring):

SACRIFICE
After body weight measurement on nursing day 4, all pups were euthanized by exsanguination under ether anesthesia.

GROSS NECROPSY
- head, chest and abdomen

Statistics:

The body weights, feed intakes, numbers of estrous images, estrous cycles, days required until copulation, gestation periods, numbers of corpora lutea, numbers of implantation marks, numbers of surviving pups and organ weights were tested by the following methods: mean value for each group ± SD, Bartlett test/F test and Dunnett’s test/Dunnett-type test/Student t-test/Aspin-Welch t-test.
The implantation index, stillbirth index, livebirth index, external abnormality index and viability index of liveborn pups were determined for each animal, and then homogeneity of variance was tested using the Bartlett test (significance level 0.01, both sides). If homogeneous, Dunnett’s test was performed, and if non homogeneous, a Dunnett-type test was performed (significance level 0.05 and 0.01, both sides).
To determine the copulation index, insemination index, conception index and delivery index, and the sex ratio of the liveborn pups, for each group the total number of animals that copulated, number of males that impregnated a female, number of pregnant females, number of females that delivered live pups, number of surviving male pups, and number of surviving female pups were determined, and the x2 test with Yates’ continuity correction was conducted (significance level 0.05 and 0.01, both sides). When a cell with expected frequency of ≤5 was observed, the test was conducted using Fisher’s direct probability method (significance level 0.05 and 0.01, both sides).

Reproductive indices:

Copulation index (%) = (number of animals that copulated/number of mated animals) x100;
Conception index (%) = (number of pregnant females/number of females that copulated) x 100;
Insemination index (%) = (number of males that impregnated a female/number of males that copulated) x 100;
Delivery index (%) = (number of females that delivered liveborn pups/number of pregnant females) x 100;
Implantation index (%) = (number of implantation marks/number of corpora lutea) x 100

Offspring viability indices:

Stillbirth index (%) = (number of stillborn pups/total number of pups born) x 100;
Livebirth index (%) = (number of liveborn pups/total number of pups born) x 100;
External abnormality index (%) = (number of pups with external abnormality/number of liveborn pups) x 100;
Sex ratio = number of male pups/(number of male pups + number of female pups);
Viability index of liveborn pups (%) = (number of surviving pups on nursing day 4/number of liveborn pups on nursing day 0) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no deaths in any administration group, and no abnormalities in general condition were observed.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout the administration period the body weights of males and females in all administration groups were equivalent to those in the control group, and no significant difference was found.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious difference in the feed intake of the males or females in the 100 mg/kg group. Females in the 300 mg/kg group had a significantly high feed intake on pregnancy day 11. In the 1000 mg/kg group, the males had a significantly high feed intake on administration day 42 and in the females had a significantly low feed intake on administration day 4 and a significantly high feed intake on pregnancy day 11. However, there was no abnormal body weight variation, and so these changes were deemed to have no toxicological significance.

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

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes attributed to test substance administration were seen in the livers and kidneys.
- Liver: Minimal brightening of hepatocytes was observed in 2/2 females in the 100 mg/kg group, 4/7 females in the 300 mg/kg group and 8/11 females in the 1000 mg/kg group. Minimal or mild centrilobular hepatocytes hypertrophy was observed in 5/5 males in the 100 mg/kg group, 10/10 males and 2/7 females in the 300 mg/kg group, and 12/12 males and 8/11 females in the 1000 mg/kg group. Minimal localized necrosis was observed in 3/11 females in the 1000 mg/kg group. - Kidney: Minimal or mild renal tubule dilatation was observed in 1/1 male in the 100 mg/kg group, 3/6 males in the 300 mg/kg group, and 12/12 males and 5/8 females in the 1000 mg/kg group. Minimal or mild renal tubule vacuolation was observed in 5/8 females in the 1000 mg/kg group. Minimal or mild renal tubule necrosis was observed in 3/8 females in the 1000 mg/kg group. Minimal or mild hyaline cast was observed in 2/6 males in the 300 mg/kg group, and 10/12 males and 1/8 females in the 1000 mg/kg group. Minimal erythrocyte cast was observed in 1/1 male in the 100 mg/kg group, 3/6 males in the 300 mg/kg group, and 6/12 males in the 1000 mg/kg group. Minimal or mild renal tubular basophilia was observed in 1/1 male in the 100 mg/kg group, 2/6 males in the 300 mg/kg group, and 12/12 males and 4/8 females in the 1000 mg/kg group. Minimal or mild renal tubule hyaline droplets was observed in 1/1 male in the 100 mg/kg group, 6/6 males in the 300 mg/kg group, and 6/12 males in the 1000 mg/kg group. Minimal or mild glomerular hyaline droplets was observed in 1/1 male in the 100 mg/kg group, 3/6 males in the 300 mg/kg group, and 12/12 males and 6/8 females in the 1000 mg/kg group. Based on the onset circumstances and histologic characteristics, the following findings in various organs and tissues were considered to be accidental changes.
- Epididymis: Mild spermatic granuloma was observed in 1 animal in the 300 mg/kg group and in 1 animal in the 1000 mg/kg group.
- Ovary: Mild follicular cyst was observed in 1 animal in the 1000 mg/kg group.
- Testis: Mild seminiferous tubular atrophy was observed in 1 animal in the control group, and moderate seminiferous tubular atrophy was observed in 1 animal in the 300 mg/kg group.
- Liver: Mild diaphragmatic hernia nodule was observed in 1 male in the control group, and minimal microgranuloma was observed in 1 female in the 1000 mg/kg group.
- Kidney: Minimal localized fibrosis was observed in 1 female in the 1000 mg/kg group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No abnormality of estrous cycle was observed in any animal. Moreover, there were no significant differences between the control group and any administration group in the number of estrous images or in mean estrous cycle duration.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was 1 pair, in the 300 mg/kg group, that did not copulate.
Copulation was confirmed in the remaining pairs within 10 days of the start of the mating period.
Moreover, there was only one infertile pairing, in the 1000 mg/kg group. No significant differences were observed between the control group and any administration group in the days required until copulation, copulation index, insemination index or the conception index. Normal delivery was achieved on pregnancy day 21.5-22.5, in all cases, and no significant differences were found in delivery index, gestation period, number of corpora lutea, number of implantation marks, implantation index, stillbirth index, the number of liveborn pups or the livebirth index. In the nursing conditions, no dams exhibited any abnormality in retrieving, nest-building or breast-feeding behaviors.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Remarks:

for reproductive toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

2

Key result

Dose descriptor:

NOEL

Remarks:

for general toxicity

Effect level:

<= 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The birth weight in females in the 100 mg/kg group was significantly high, but this was not doserelated, and the change was not toxicologically significant. There were no other significant differences between the control group and any administration group.

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

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thymic remnant in the neck were observed in 1 male in the control group, 1 male in the 100 mg/kg group, 1 male in the 300 mg/kg group, and 1 male and 3 females in the 1000 mg/kg group; this change occurred at low frequency, and, based on the developmental characteristics, was thought to be an accidental change. In addition, situs inversus totalis was observed in 1 female in the control group.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Remarks:

for developmental toxicity

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance was less than 100 mg/kg/day in males and females, and that the no-observed effect dose with respect to reproduction and development toxicity was 1000 mg/kg/day in male and female parents and pups.

Executive summary:

The test substance was administered at 0 (control group: aqueous 0.5 w/v% carboxymethyl cellulose solution with 0.1 v/v% Tween® 80 added), 100, 300 and 1000 mg/kg to Sprague- Dawley SPF rats, for 14 days before mating and throughout the mating period until the day before autopsy in males (42 days), and for 14 days before mating and throughout the mating period and gestation period until day 4 of nursing in females (42-48 days), and the repeated dose toxicity and reproductive and developmental toxicity were investigated according to a OECD TG 421 study. In the parent animals, test substance administration had no observable effect on estrous cycle, days required until copulation, copulation index, insemination index or conception index. Furthermore, test substance administration had no observable effect on delivery index, gestation period, number of corpora lutea, number of implantation marks, implantation index, stillbirth index, number of liveborn pups, livebirth index or sex ratio, and no abnormalities in nursing condition were observed. Therefore, test substance administration was thought to have had no effect on reproductive functions such as copulation ability in males or females, or on insemination ability or conception ability, or on pregnancy maintenance, delivery and nursing behavior, etc. of dams, even in the 1000 mg/kg group. In the liveborn pups, no changes due to test substance administration were observed in body weight at birth or on day 4 of nursing, in males or females, and no changes due to test substance administration were observed on external examination at birth, or in the autopsy findings on day 4 of nursing, or in the viability index. It was determined from these results that the no-observed effect dose with respect to the repeated administration toxicity of the test substance in males and females was less than 100 mg/kg/day, and that the no-observed effect dose with respect to reproduction and development toxicity in male and female parents and pups was 1000 mg/kg/day.