Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Study:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Weight of evidence information based on information of various test chemical.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
The above experiments were performed to assess the effects of the test chemical on the reproduction parameters of the test animals.
GLP compliance:
not specified
Limit test:
no
Justification for study design:
No Data Available
Specific details on test material used for the study:
- Molecular weight (if other than submission substance): 256.343 g/mol
- Substance type: Organic
- Physical state: White Powder
- Impurities (identity and concentrations): No Data Available
Species:
other: Study 1: no data; Study 2 and 3: rat; Study 4: mouse; Study 5 and 6: rat
Strain:
other: Study 1: no data; Study 2: Crj:CD (SD) IGS; Study 3: Fischer 344/DuCrj; Study 4: Crj:CD-1 (ICR); Study 5 and 6: Wistar
Details on test animals or test system and environmental conditions:
Study 2: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 10 week old
- Weight at study initiation: 332-383 g for male, 206-238 g for female.
- Fasting period before study: Not available
- Housing: Stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.
- Diet (e.g. ad libitum): The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.)
- Water (e.g. ad libitum): The drinking water was freely taken in all of the tap water
- Acclimation period: Not available
ENVIRONMENTAL CONDITIONS .
- Temperature (°C): 20 to 26 ° C.
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 times / hour.
- Photoperiod (hrs dark / hrs light): Light and dark each for 12 hours (lighting: 6 am to 6 pm)

Study 3: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4-week-old
- Weight at study initiation: 57.7-70.7 g
- Fasting period before study: No data available
- Housing: Animals were housed individually in chip-bedded plastic and stainless steel suspended cages in a controlled environment.
- Diet (e.g. ad libitum): The standard chow diet CE-2
- Water (e.g. ad libitum): No data available
- Acclimatization period: No data available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 11-hrs light/13-hrs dark

Study 4: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 4-week-old
- Weight at study initiation: 19.3-22.8 g
- Fasting period before study: No data available
- Housing: Animals were housed individually in chip-bedded plastic and stainless steel suspended cages in a controlled environment.
- Diet (e.g. ad libitum): The standard chow diet CE-2
- Water (e.g. ad libitum): No data available
- Acclimatization period: No data available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 11-hrs light/13-hrs dark

Study 5: TEST ANIMALS
- Source: SLC Co. (Hamamatsu, Japan)
- Age at study initiation: 4 weeks old
- Weight at study initiation: No data avilable
- Fasting period before study: No data available
- Housing: Animals were housed individually in aluminum hanging cages.
- Diet (e.g. ad libitum): Basal diet powder F-2 (Funabashi Farm, Japan)
- Water (e.g. ad libitum): No data available
- Acclimatization period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24±1°C
- Humidity (%):55±5%
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-hrs light/12-hrs dark


Route of administration:
oral: feed
Details on exposure:
Study 2: PREPARATION OF DOSING SOLUTIONS: The test chemical was prepared by suspending it in a 5% gum arabic solution. The test chemical was converted to purity, and the dose was expressed in terms of the original weight. Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation. Also, as a result of confirming the concentration of the test chemical in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test chemical.
DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A
VEHICLE
- Justification for use and choice of vehicle (if other than water): 5% gum arabic
- Concentration in vehicle: 12.5, 50, 200 or 800 mg/kg/day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Study 3: PREPARATION OF DOSING SOLUTIONS:
The experimental pellet diets were made from powdered CE-2 and MM using a mixer, a pellet maker and a dryer. The diets were kept in a refrigerator at 10°C before use. The basal CE-2 diet (crude protein: about 25%) was free of contamination by pesticides.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Basal diet CE-2
- Storage temperature of food: At 10°C
VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet CE-2
- Concentration in vehicle: 0 or 0.06% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): E2070-UZ; E2063-T8; E2034-YA and E2104-OX.
- Purity: No data available

Study 4: PREPARATION OF DOSING SOLUTIONS:
The experimental pellet diets were made from powdered CE-2 and MM using a mixer, a pellet maker and a dryer. The diets were kept in a refrigerator at 10°C before use. The basal CE-2 diet (crude protein: about 25%) was free of contamination by pesticides.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): Basal diet CE-2
- Storage temperature of food: At 10°C
VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet CE-2
- Concentration in vehicle: 0 or 0.25% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): E2070-UZ; E2063-T8; E2034-YA and E2104-OX.
- Purity: No data available

Study 5: PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed into the basal diet powder F-2 prior to pelleting at the desired concentrations.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): F-2 basal diet powder.
- Storage temperature of food: At 4°C until use.
VEHICLE
- Justification for use and choice of vehicle (if other than water): F-2 basal diet powder
- Concentration in vehicle: 0, 0.01, 0.03 or 0.1% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Study 6: PREPARATION OF DOSING SOLUTIONS:
The test chemical was mixed into the basal diet powder F-2 prior to pelleting at the desired concentrations.
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): F-2 basal diet powder.
- Storage temperature of food: At 4°C until use.
VEHICLE
- Justification for use and choice of vehicle (if other than water): F-2 basal diet powder
- Concentration in vehicle: 0, 0.12, 0.6 or 3.0% per day
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on mating procedure:
Study 2: - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy vaginal plug
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. Not available
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not available
- After successful mating each pregnant female was caged (how):Not available
- Any other deviations from standard protocol:Not available

Study 3,4,5 and 6: Mating was not performed

Details on analytical verification of doses or concentrations:
Study 2,3,4,5 and 6: No Data Available
Duration of treatment / exposure:
Study 2: Male: 50-52 days
Female: 40 to 48 days.

Study 3: 2 months

Study 4: 2 months

Study 5: 18 months

Study 6: 12 weeks
Frequency of treatment:
Study 2, 3, 4,5 and 6: Daily
Details on study schedule:
Study 2,3 and 5: No data available

Study 5: - Dose selection rationale: Because of the severe histopathological changes in the testis of the lowest-dose group (0.12% per day in the diet) males in the 12-week study, doses selected for the 18-month study were 0, 0.01, 0.03 and 0.1% per day in the diet.
Male and female Wistar rats were fed 0, 100, 300 and 1000 ppm of the test substance in diet for 18 months .Various examinations were done during and after the exposure period.

Study 6: Male and female Wistar rats were fed 0,0.12, 0.6 or 3.0% per day of the test substance in diet for 12 weeks .Various examinations were done during and after the exposure period.
Remarks:
Study 2: Doses / Concentrations:
12.5, 50, 200, 800 mg/kg/day (in 5% gum arabic)
Basis:
no data

Study 3: Doses / Concentrations:
0.06% per day (ca. 38.6-58.0 mg/kg bw/day)
Basis:
nominal in diet

Study 4: Doses / Concentrations:
0 or 0.25% per day (ca. 371-457mg/kg bw/day)
Basis:
nominal in diet

Study 5: Doses / Concentrations:
0, 0.01, 0.03 or 0.1% per day
Basis:
nominal in diet

Study 6: Doses / Concentrations:
0,1200,6000,30000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Study 2: Control-12 female and 12 male
12.5 mg/kg/day- 12 female and 12 male
50 mg/kg/day-12 female and 12 male
200 mg/kg/day-12 female and 12 male
800 mg/kg/day-12 female and 12 male

Study 3: Control: 8 males
0.06% per day: 8 males

Study 4: Stduy 4: Control-8 male mice
0.25%-8 male mice

Study 5: Control: 30 males, 30 females
0.01% per day: 30 males, 30 females
0.03% per day: 30 males, 30 females
0.1% per day: 30 males, 30 females

Study 6:Control-10 male and 10 female
1200 ppm-10 male and 10 female
6000 ppm-10 male and 10 female
30000 ppm-10 male and 10 female
Control animals:
other: Study 2,3,4,5: Yes, concurrent; Study 6: Yes, plain diet
Details on study design:
Study 2: No data Available

Study 3: No Data Available

Study 4: Feeding Experiment:Male mice were fed 0.25% of the test substance in diet for 2 months,followed by various examinations.

Study 5: No data available

Study 6: Male and female Wistar rats were fed 0(control), 0.12, 0.6 or 3.0% of the test substance in diet for 12 weeks .Various examinations were done during and after the exposure period.
Positive control:
Study 2,3,4,5 and 6: No Data Available

Parental animals: Observations and examinations:
Study 2: Males:
Clinical Signs: The general condition and the presence or absence of death were observed twice a day before and after administration.

Study 3: Clinical signs of toxicity were recorded daily. Body weight and food consumption were occasionally measured.
Body weights: Body weight was measured twice a week.
Food Consumption: Food consumption was measured twice a week from 14 days before the start of the mating and after the end of the mating cycle.
Necropsy and Histopathology: On the day after the final administration, the animals were sacrificed by abdominal aorta under ether anesthesia, necropsied, and the tail weight of testis, epididymis and epididymis was measured. The relative weight was also calculated by dividing each organ weight by the final body weight. Testis and epididymis heads were fixed in Bouin's solution. Prostate and seminal vesicles were fixed in 20% neutral buffered formalin. Testis and epididymis were paraffin-embedded specimens prepared according to a conventional method. HE stained tissue specimens were prepared for the control group and the testis and epididymis heads in the 800 mg / kg group, and seminal vesicles with abnormality at necropsy (6 subjects in the 800 mg / kg group), and histopathology An inspection was carried out. For testes and epididymis heads considered to be different in number of animals showing abnormalities compared with the control group in the 800 mg / kg group examination, the 12.5, 50 and 200 mg / kg group was also examined did.
Sperm paramters: The tail of the right epididymis was divided in sperm culture (0.5% bovine serum albumin plus Medium 199) warmed to 37 ° C. and used as a sperm stock solution. The sperm concentrate was used to examine sperm activity, sperm viability and sperm morphology.Sperm activity was determined by diluting the sperm diluted solution with sperm culture solution in sperm culture solution and incubating the sperm diluted solution with HTM-IVOS (Hamilton Thorne Research) after incubating for about 30 minutes (culture conditions: 37 ° C., 5% carbon dioxide, 95% The active spermatozoa ratio was obtained and the reference point moving speed, the shortest moving speed, the total moving speed and the number of crossings of the head were calculated for the active sperm.
As the viability of sperm, according to the method 2) of Kato et al., The sperm stock solution was diluted about 3 times with the sperm culture solution in the microwell plate, then 16 μM calcein acetoxy methyl ester and 8 μM ethidium homodimer-1 After incubation and staining for about 2 hours (culture conditions: 37 ° C, 5% carbon dioxide, 95% air), spermatozoa were classified as living sperm, mid-dead sperm and killed sperm under a fluorescence microscope and surviving sperm ratio and survival The sperm ratio was determined. For survival, mid-death, and death sperm determination, green fluorescent color development is observed in the head to tail area, living spermatozoa, red fluorescent coloring in the head and green fluorescent coloring in the tail are observed in the head Somewhat dead sperm, sperm on the head, red fluorescent coloring was observed in the head, but those that did not show fluorescent coloring in the tail were regarded as dead sperm.
The morphology of the sperm was prepared by mixing a sperm stock solution and a 10% eosin stain solution, and smears were prepared and observed under a microscope.
Sperm counts were calculated using HTM-IVOS after homogenizing the left epididymis tail. The number of spermatozoa per 1 g of the tail of the left epididymis was also calculated.
Females:
Clinical Signs: The general condition and the presence or absence of death were observed twice a day before and after administration.
Sex cycle: The sex cycle was observed once a day from the administration start date to the mating confirmation date. In addition, when , the estrus period was observed over 2 consecutive days it was counted as 1 time.
Body Weights: Body weights were measured twice a week during the 14 days before the mating and during the mating period, at 0, 7, 14 and 21 gestation during gestation, on 0 and 4 days of feeding during the feeding period, respectively .
Food Intake: Food consumption was measured twice a week until 14 days before the start of the mating. Also, during pregnancy, gestation was measured on 2, 9, 16 and 21 gestation, and 4 days after nursing during gestation period.
Delivery Status: Mating females were allowed to spontaneously deliver, and the presence or absence of abnormality in labor condition and confirmation of end of delivery were confirmed once a day from day 21 of pregnancy until 25th day of pregnancy. If delivery was completed at 10:00 am, that day was taken as nursing day 0. Females who did not deliver until 25th day of pregnancy were necropsied after lethality from the abdominal aorta under ether anesthesia.
Necropsy and Histopathology: Maternal animals were observed for nursing condition once a day until 4th day of nursing and autopsied after death from the abdominal aorta under ether anesthesia on the day when all newborn cases died or on nursing 4th day, necropsied, and the number of implantation traces and I counted the corpus luteum. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight. For the ovary, a paraffin-embedded specimen was prepared according to an ordinary method. HE stained tissue specimens were prepared for the control group and the ovaries of the 800 mg / kg group and histopathological examination was performed.

Study 4:Clinical signs of toxicity were recorded daily. Body weight and food consumption were occasionally measured.

Study 5 and 6: Clinical signs were recorded daily. Body weight and food consumption were monthly.

Oestrous cyclicity (parental animals):
Study 2: the estrus period was observed over 2 consecutive days

Study 3,4,5 and 6: No data available
Sperm parameters (parental animals):
Study 2: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, viability, sperm morphology

Study 3: Testicular sperm content and daily sperm production were determined.

Study 4: Testicular sperm content and daily sperm production were determined.

Study 5 and 6: No data available
Litter observations:
Study 2: Number of pups, live birth index, body weights of both sexes, number of stillbirths.
Study 3,4,5 and 6: No data available
Postmortem examinations (parental animals):
Study 2: Yes, in males On the day after the final administration, the animals were sacrificed by abdominal aorta under ether anesthesia, necropsied, and the tail weight of testis, epididymis and epididymis was measured. The relative weight was also calculated by dividing each organ weight by the final body weight. Testis and epididymis heads were fixed in Bouin's solution. Prostate and seminal vesicles were fixed in 20% neutral buffered formalin. Testis and epididymis were paraffin-embedded specimens prepared according to a conventional method. HE stained tissue specimens were prepared for the control group and the testis and epididymis heads in the 800 mg / kg group, and seminal vesicles with abnormality at necropsy (6 subjects in the 800 mg / kg group), and histopathology An inspection was carried out. For testes and epididymis heads considered to be different in number of animals showing abnormalities compared with the control group in the 800 mg / kg group examination, the 12.5, 50 and 200 mg / kg group was also examined. In females, Maternal animals were observed for nursing condition once a day until 4th day of nursing and autopsied after death from the abdominal aorta under ether anesthesia on the day when all newborn cases died or on nursing 4th day, necropsied, and the number of implantation traces and I counted the corpus luteum. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight. For the ovary, a paraffin-embedded specimen was prepared according to an ordinary method. HE stained tissue specimens were prepared for the control group and the ovaries of the 800 mg / kg group and histopathological examination was performed.

Study 3: Hematological and serum biochemical examinations were conducted after 16 hrs starvation. All animals were studied for histological changes.

Study 4: At termination of administration, mice were killed and blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys and liver were dissected out and weighed. Testis was fixed with formalin, sectioned, stained routinely with hematoxylin and eosin and observed microscopically.

Study 5: Hematological and serum biochemical examinations were conducted after 16 hrs starvation at month 6, 12 and 18. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg).
At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examionation.

Study 6: Hematological and serum biochemical examinations were conducted after 16 hrs starvation at week 4 and 12. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg).
Urinalysis was also conducted in fresh urine for pH, protein, glucose, ketone bodies, occult blood, bilirubin and urobilin in the morning at weeks 4 and 12.
At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examionation.
Postmortem examinations (offspring):
Study 2: The surviving pups was sacrificed by abdominal aorta from the abdominal aorta under necropsy under ether anesthesia on 4th day of nursing and then necropsied.

Study 3, 4, 5 and 6: No data available
Statistics:
Study 2: For statistical analysis, homodispersity was tested by the Bartlett method. In the case of equi-variance, variance analysis was performed by one-way method, and if significant, it was performed by the Dunnett method. On the other hand, when it was not recognized as equal variance, we performed analysis by the one-way method using rank order (Kruskal-Wallis test), and if significant, we used Dunnett type test method using ranking. Mating rate, conception rate and birth rate were determined by χ ^ 2 test.
In histopathological examination, the toxicological effects were suggested in the 800 mg / kg group, and findings of organs and tissues examined for other groups were ranked using comparison between groups with the control group Dunnett type test method was used. Therefore, when a significant difference was observed with the control group, the dose reactivity test was conducted using Cochran · Armitage trend test.

Study 3 and 5 and 6: All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data.

Study 4: Variations of body and organ weights are routinely expressedas per standard deviation. Bartlett’s test, analysis of variance (ANOVA), Kruskal–Wallis test, and Dunnett’s parametric or non-parametric test were used as tests for significance of differences. Fisher’s exact test was used for histopathologic data. The limit for significance was set at P<0.05.
Reproductive indices:
Study 2: Implantation Index, Resorption Index, Mating Index, Gestational Index
Study 3,4,5 and 6: No data available
Offspring viability indices:
Study 2: Pups viability index, Litter Index
Study 3,4,5: No data available
Study 6: Survival indices at wk 12
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Study 2: In males, transient salivation was observed in the 200 and 800 mg / kg group in general condition observation. In addition, loose stools were observed in each group including the control group, but no relation with the dose was observed.

Study 3: No effects observed

Study 4: No data available

Study 5: No effects observed

Study 6: Haemorrahage from the nasal cavity was observed from all the dead animals. Diarrhea was observed in two of the four of 3.0% of the males and one in four females from 3.0% dose group of dead animals.
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
Study 2,3,4,5: No data available
Mortality:
no mortality observed
Description (incidence):
Study 2: No Mortality was observed in either of males and female animals of treated groups.
Study 3,4,5: No data available
Study 6: Mortality was observed in males of 0.6% and 3.0% dosed group and females of 3.0% dose group from weeks 3 to 4.
Body weight and weight changes:
not specified
Description (incidence and severity):
Study 2: In males, there was no significant difference in body weight at any measurement day in each administration group compared with the control group. In females, Before the mating, there was no significant difference in body weight at any measurement day in each administration group compared with the control group.
During the gestation period, there was no significant difference in body weight at any measurement day in the 12.5, 50 and 200 mg / kg group compared with the control group. In the 800 mg / kg group, there was a significant lower value of body weight on 0 to 21 days of pregnancy than in the control group.
During the nursing period, there was no significant difference in body weight at any measurement day in the 12.5 and 50 mg / kg group compared to the control group. In the 200 and 800 mg / kg group, a significant lower value of body weight was seen on 4th day of nursing compared with the control group.

Study 3: There were no significant differences in body weights among F344 rats administered with the test chemical at a level of 0.06% per day.

Study 4: Body weight gain was not significantly depressed in male mice fed with the test chemical.

Study 5: effects observed treatment related

Study 6: In both the sexes, severe suppression was observed in 0.6 and 3.0 % dose groups.
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
Study 2: In males, in the 12.5, 50 and 200 mg / kg groups, there was no significant difference in food intake on any measurement day compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. In females, Before the mating, there was no significant difference in food consumption on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 mg / kg group, a significant lower value of food intake was observed on days 3 and 6 compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. During the gestation period, there was no significant difference in food intake on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 and 800 mg / kg group, a significant lower value of food intake was seen on the 2nd day of pregnancy than in the control group. There was no significant difference in food intake in the 12.5 and 50 mg / kg group during the nursing period compared with the control group. In the 200 and 800 mg / kg group, a significant low value of food intake was seen on the 4th day of nursing compared with the control group.

Study 3: There were no significant differences in body weights among F344 rats administered with the test chemical at a level of 0.06% per day.

Study 4: No effects observed

Study 5: effects observed treatment related

Study 6: Decreased mean food intake was observed in males of 0.6 and 3.0% dose group and females of 3.0% dose group.
Food efficiency:
not specified
Description (incidence and severity):
Study 2,3,4,5 and 6: No data available

Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
Study 2,3,4,5 and 6: No data available

Ophthalmological findings:
not specified
Description (incidence and severity):
Study 2,3,4,5 and 6: No data available

Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Study 2,3,4,5: No data available

Study 6: Significant decrease in the RBC was observed in 0.6% males at week 12, but not in treated females. Significant decrease in HGB at 3.0% dose group males in week 4, and 0.6% dose group males and all treated females at week 12. Although, not significant, a decrease in PLT in both the sexes of 0.3% were observed in week 12.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Study 2,3,4,5: No data available

Study 6: Significant decrease in GLU was observed in 3.0% dose groups males and 0.6 and 3.0% dose group females at week 12. PL was significantly increased in 0.6 and 3.0% males and 0.6% females at week 4 and 0.6% and 3.0% dose group females at week 12. T-CHO and F-CHO were significantly elevated elevated in both the sexes at 0.6 and 3.0% dose groups in week 4 and thereafer. CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% at week 4 and in 0.6% and 3.0% females at week 12. Gamma-GTP was significantly increased in 0.6 and 3.0% females at week 4 and 0.6% males and 0.6 and 3.0% females at week 12.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Study 2,3,4,5: No data available

Study 6: There were no changes observed in biochemical urine parameters from the test chemical treated rats.
Behaviour (functional findings):
not specified
Description (incidence and severity):
Study 2,3,4,5 and 6: No data available
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Study 2,3,4,5 and 6: No data available

Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In males, In the testes, the atrophy of seminiferous tubules was observed in 6 patients in the 200 mg / kg group and in 12 cases in the 800 mg / kg group. One case of degeneration of seminiferous tubules was observed in the 200 mg / kg group. A reduction in sperm was observed in the 200 mg / kg group in one case. Two cases of giant cells appeared in 50 mg / kg group and two cases in 200 mg / kg group. In the epididymis, nine cases of sperm decrease in 200 mg / kg group and 12 cases in 800 mg / kg group were observed. In the testes, atrophy of seminiferous tubules and spermatozoa in epididymis was significantly different from the control group in the 200 and 800 mg / kg group, and the dose correlation was also confirmed. No abnormalities were found in the seminal vesicle (at 6 months in the 800 mg / kg group) who showed atrophy at necropsy. In females, in the ovary, no abnormalities were observed in the control group and the 800 mg / kg group.

Study 3: Vacuolation of Sertoli cells, exfoliation, retention of elongated spermatids in the basilar region of a stage X, degeneration of spermatids, disappearance of basement membrane, and broken tails of elongated spermatids were, however, observed in the test chemical treated group.

Study 4: Sloughing of seminiferous tubules was found in 50% mice. Giant cells were abundantly observed in 75% mice. In addition, dilatated lumen and Leidig cells vacuolization was also observed.

Study 5: effects observed treatment related

Study 6: In male rats, testicular atrophy and appearance of the giant cells were observed in the 0.6 and 3.0% dose groups in week 4. At week 12, testicular atrophy was observed and pronounced at all the dose groups and all the male treated animals. Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed. In females, atrophy of the ovaries and uterus were apparent in the 0.6% and 3.0% groups. In both the sexes, thymus atrophy and bone marrow hypoplasia were noted in the 0.6 and 3.0% groups. No histopathological changes were observeds in any other organs.
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
Study 2,3,4,5 and 6: No data available
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Study 2,3,4 and 6: No data available

Study 5: Effects observed treatment related
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Study 2: There was no significant difference in the number of estrus counts during the pre-mating period (14 days) compared with the control group in each administration group.

Study 3,4,5 and 6: No data available

Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 mg / kg group, compared with the control group, there were no significant differences in activity sperm ratio, reference point moving speed, shortest distance traveling speed, crossing number of head crossings, malformed sperm rate, survival sperm rate, surviving sperm rate, sperm count, epididymis There was no significant difference in sperm count per 1 g of tail. In the 12.5 mg / kg group, a significant increase in the total migration rate was observed compared to the control group, but it was not a change related to the dose, the other items related to sperm activity (activity sperm ratio, The reference point moving speed, the shortest moving speed, and the number of crossings of the head), it was judged that it was not due to administration. In the 50 mg / kg group, there was a significant lower value of sperm count per g of active sperm ratio, survival sperm ratio, survival sperm ratio, sperm count and epididymal tail compared with control group, significant high value of malformed sperm ratio Was observed. In the 50 mg / kg group, a significant high value of the crossing number of the head was seen compared to the control group, but because it was not a change related to the dose, it was judged that it was not due to administration. In the 200 mg / kg group, there was a significant lower value of active sperm ratio, survival sperm ratio, survival sperm ratio, sperm count and sperm count per gram of epididymal tail compared to control group. In the 200 mg / kg group, the active spermatozoa rate in 6 cases was 0%, but no significant difference was found in the calculated animal's reference point moving speed, shortest distance moving speed and total moving speed. In the 200 mg / kg group, there was a significant lower value of the crossing number of the head compared to the control group, but it was very slight difference. In the same group, seven cases where sperm morphology observation was possible showed a significant high value of malformed sperm ratio as compared with the control group. Significant lower values ​​of sperm count and sperm count per gram of epididymal body tail were observed in the 800 mg / kg group compared to the control group. In the 800 mg / kg group, the active spermatozoic rate was 0% in all animals, a significant difference was recognized as compared with the control group, and the reference point moving speed, the shortest moving speed, the total moving speed and the crossing number of the head Can not be measured. In the 800 mg / kg group, only one animal was able to observe morphology of spermatozoa, but most of it was malformation sperm. In the same group, observable spermatozoa could not be obtained, so it was not possible to calculate surviving sperm ratio and survival sperm ratio.

Study 3: Daily sperm production (DSP) and DSP/g testis were significantly decreased in the test chemical-treated rats.

Study 4: No effects observed

Study 5: No data available

Study 6: Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed.
Reproductive performance:
not specified
Description (incidence and severity):
Study 2: There was no significant difference in the number of estrus counts during the pre-mating period (14 days) compared with the control group in each administration group. Since mating was confirmed in all cases used for mating, the mating rates of each group were all 100%. There was no significant difference in the number of days required for copulation between the control group and each administration group. One unfertilized female was found in the 200 mg / kg group, but no significant difference was observed in the conception rate between each administration group and the control group.

Study 3,4,5 and 6: No data available

Study 2: Male
At 50 mg/kg/day:
Giant cell formation in the testis. Decrease in sperm motility ratio and number of sperms in the cauda epididymis. Increase in abnormal sperm ratio.
At 200 mg/kg/day:
Atrophy of the testis and the epididymis. Decrease in the absolute and relative testis and epididymis weights. Atrophy of seminiferous tubules. Degeneration of seminiferous tubules. Decrease in number of sperm in the cauda epididymis. Giant cell formation.
At 800 mg/kg/day:
Transient decrease in food consumption. Atrophy of the testis, the epididymides and the seminal vesicle. Decrease in the absolute and relative testis and epididymis weights. Atrophy of seminiferous tubules in the testis. Observation of no motile sperm. Increase tendency in number of abnormal sperm. Decrease in number of sperm in the cauda epididymis.
Female
At 200 mg/kg/day:
Suppression of body weight gain during the lactation period. Lower food consumption during pre-mating, pregnancy and lactation periods.
At 800 mg/kg/day:
Suppression of body weight gain during the pregnancy and the lactation periods. Lower food consumption during pre-mating, pregnancy and lactation periods..

Study 3: No significants differences in body weight gain compared to control, no effects on the absolute organ weights, decrease in relative testicular and epididymal weights, no weights of other sex accessory organs were reduced, DSP/g testis were significant decreased, no significant change in serum testosterone level.
Histopathological findings:
Vacuolation of Sertoli cells ,exfoliation ,retention of elongated spermatids in the basilar region of stage X ,degeneration of spermatids, giant cells ,multinucleated giant cells , disappearance of basement membrane , broken tails of elongated spermatids , necrotic spermatocytes and/or spermatogonia,disappearance of germ cells , disappearance of round spermatids were observed.
ERalpha competitive binding of the test chemical:
ER alpha competitive binding of the test chemical showed that the test substance did not inhibit E2-ER alpha binding (up to 10-3 M).Serum testosterone levels were not significantly changed after treatment with the test chemical.

Study 4: No absolute sex accessory organ weights and liver and kidney weights were changed in mice fed the test substance.Sloughing of seminiferous tubules,Giant cells,Dilatated lumen,Leidig cells vacuolization.
ERalpha competitive binding of the test chemical in vitro:
the test substance did not inhibit E2 -ERalpha binding (up to 10-3M)
Serum testosterone levels were not significant changed after treatment

Study 5: Clinical signs and mortality: No remarkalbe changes in general appearance were observed in any rat. survival rates in all treated animals were comparable to those of control (see Table below).
Body weight: Significant suppression of body weight gain was only observed in 0.1% per day males from month 6 and 0.1% per day females from month 1.
Mean food intakes per rat or per kg BW in all treated groups were comparable to those of controls.Mean efficiency of feed utilization was dose-dependently decreased in both sexes.
Organ weights (testis): Significant increases or tendencies for increase in relative liver weights were observed in the 0.1% per day animals of both sexes.
Significant decreases in absolute and relative testis weights were observed in the 0.1% group throughout the study. In contrast, no change in ovary weights was observed in any of the treated females. No changes in other organs were observed in any treated groups for either sex.
Histopathology (incidence and severity):
Histopathological lesions were only observed in the testis and epididymis of males.Atrophy of testicular tubules and spermatogenic arrest and epididymis hypospermia were observed limited to the 0.1% group, throughout the study. However, no interstitial cell tumors were apparent.
In the other organs of males, no changes induced by MBMBP were observed and no changes in any organs were apparent in females.
No neoplastic lesions which could be attributed to MBMBP were observed in any organs of either sex.
Male: Atrophy of testicular tubules, spermatogenic arrest and epididymis hypospermia were observed in the 1000 ppm group.
Female: No significant effect was observed.
Haematology:In the hematological and serum biochemical analyses, several parameters demonstrated significant alteration. However, none appeared to be of biological significance, since they did not show the same tendency throughout the experimental period and/or the degrees of change were very small.
Gross pathology incidence and severity: Organ weight changes:
Male: Increase in liver weight at 300 ppm (absolute (p<0.05) and relative (p<0.01)); decrease in testis weight at 300 ppm (absolute and relative) (p<0.01).
Female: Increase in liver weight at 1000 ppm (relative) (p<0.01). no changes in ovary weights were observed in any of the treated females. no changes in other organs were observed in any treated groups for males and females.

Study 6: Clinical signs :Deaths were observed in the 0.6 and 3.0% per day in males and in 3.0% females from weeks 3 to 4. All mortalities was accompanied by hemorrhage from the nasal cavity. Diarrhea was observed in two of the four of the 3.0% per day males and in one of the four 3.0% per day females which died.
Body weight and food consumption:In both sexes, severe suppression of body weight gain was observed when treated with 0.6 and 3.0% per day. Decreases of mean food intake per rat were observed for the 0.6 and 3.0% per day males and for the 3.0% per day females.
Organ weights:Relative liver weights were dose-dependently increased in males and relative and absolute liver weights were dose-dependently increased in females. Absolute and relative testis weights were time- and dose-dependently decreased in all treated males. In females, absolute and relative ovary weights were significantly decreased at 3.0% per day at weeks 4 and 12.
Histopathology:In male rats, testicular atrophy and the appearance of giant cells were observed at 0.6 and 3.0% per day at week 4. At week 12, testicular atrophy was pronounced and observed in all-treated rats. Decrease of spermatogenesis was evident in all treated males at week 4 and thereafter. Interstitial edema in the testis was also apparent in all treated rats at week 12. At weeks 4 and 12, epididymis atrophy and hypospermia in the 3.0% per day males and atrophy of the seminal vesicles and prostate at 0.6% and 3.0% per day were observed.
In females, atrophy of the ovaries and uterus were apparent in the 0.6 and 3.0% per day groups. In both sexes, thymus atrophy and bone marrow hypoplasia were noted in the 0.6 and 3.0% per day groups.
No histopathological changes were observed in any other organs.
Hematology
Significant decrease in the RBC was observed in 0.6% per day males at week 12, but not in treated females. Significant decrease of HGB was observed in 3.0% per day males at week 4, and 0.6% per day males and all treated females at week 12. Although not significant, a decrease of PLT in both sexes of the 0.3% per day group noted at week 12.
Clinical chemistry
Significant decrease of GLU was observed in 3.0% per day males and 0.6 and 3.0% per day females at week 4 and 3.0% per day females at week 12. PL was significantly increased in 0.6 and 3.0% per day males and 0.6% per day females at week 4 and 0.6 and 3.0% per day females at week 12. T-CHO and F-'CHO were significantly elevated in both sexes of the 0.6 and 3.0% per day groups at week 4 and thereafter.
CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% per day at week 4 and in 0.6 and 3.0% per day females at week 12. 7-GTP was significantly increased in the 0.6 and 3.0% per day females at week 4 and the 0.6% per day males and 0.6 and 3.0% per day females at week 12.
There were no changes in the biochemical parameters for urine from MBMBP treated rats.

Dose descriptor:
NOAEL
Effect level:
12.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed organ weights,Sperm parameter.
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decrease in sperm motility ratio and number of sperms in the epididymis cauda, increase in abnormal sperm ratio
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed on Number of pups, live birth index, body weights of both sexes, number of stillbirths.
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased body weight gain, number of corpora lutea, number ofimplantation scars and number of pup born and lower food consumption.
Dose descriptor:
LOAEL
Effect level:
58 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathological changes in testis and reduces sperm production.
Dose descriptor:
LOAEL
Effect level:
457 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathological changes in testis
Dose descriptor:
NOAEL
Effect level:
4.2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed.body weight; food consumption and compound intake; food efficiency; water consumption and compound intake; gross pathology; organ weights; histopathology.
Dose descriptor:
LOAEL
Effect level:
12.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathological changes in testis and effects on spermatogenesis, and absolute liver weight and body weight.
Dose descriptor:
NOAEL
Effect level:
15.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed. body weight; food consumption and compound intake; food efficiency; water consumption and compound intake; gross pathology; organ weights; histopathology.
Dose descriptor:
LOAEL
Effect level:
88 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Histopathological changes in the testis, decrease in testis weight, biochemical changes.
Dose descriptor:
NOAEL
Effect level:
104 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed except slight decrease in hemoglobin content.
Dose descriptor:
LOAEL
Effect level:
618 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Death, decreased body weight, increased liver weights and histopathological changes in ovary and uterus.
Critical effects observed:
not specified
Organ:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
Study 2: There was no abnormality in each group in the observation of the external table of the newborn. Also, there was no abnormality in each group in the general condition of the newborn.

Study 3 and 6: No data available
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
Study 2: No data available

Study 3 and 6: No data available
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed.

Study 3 and 6: No data available
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference.

Study 3 and 6: No data available
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
Study 2, 3 and 6 No data available

Food efficiency:
not specified
Description (incidence and severity):
Study 2, 3 and 6: No data available

Water consumption and compound intake (if drinking water study):
not specified
Description (incidence and severity):
Study 2,3 and 6: No data available

Ophthalmological findings:
not specified
Description (incidence and severity):
Study 2, 3 and 6: No data available

Haematological findings:
not specified
Description (incidence and severity):
Study 2, 3 and 6: No data available

Clinical biochemistry findings:
not specified
Description (incidence and severity):
Study 2, 3 and 6: No data available

Urinalysis findings:
not specified
Description (incidence and severity):
Study 2, 3 and 6: No data available

Sexual maturation:
not specified
Description (incidence and severity):
Study 2, 3 and 6 No data available

Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
Study 2, 3 and 6 No data available
Gross pathological findings:
not specified
Description (incidence and severity):
Study 2: No abnormality was found in any of the groups.

Study 3 and 6: No data available
Histopathological findings:
not specified
Description (incidence and severity):
Study 2, 3 and 6 No data available
Other effects:
not specified
Description (incidence and severity):
Study 2, 3 and 6 No data available
Behaviour (functional findings):
not specified
Description (incidence and severity):
Study 2, 3 and 6 No data available
Developmental immunotoxicity:
not specified
Description (incidence and severity):
Study 2 and 6: No data available
Study 2: There was no abnormality in each group in the observation of the external table of the newborn. Also, there was no abnormality in each group in the general condition of the newborn. In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed. In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference. No abnormality was found in any of the groups.

Study 3: No data available

Study 4: Not examined/No data

Study 6: No data available
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effect level is estimared
Remarks on result:
other: Not measured/tested
Remarks on result:
other: not
Critical effects observed:
not specified
System:
other: Not specified
Critical effects observed:
not specified
System:
other: not specified
Reproductive effects observed:
not specified
Treatment related:
not specified
Conclusions:
Based on all the observation and results, it was concluded that the NOAEL for the test chemical was considered to be 50 mg/kg bw.
Executive summary:

Reproductive Toxicity Study:

Study 2:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Study 3:

In a two month feeding study with male F344/Crj (Fischer) rats, the test chemical was administer in F344/Crj (Fischer) rats. The rats were housed individually in chip-bedded plastic and stainless steel suspended cages, respectively, in an air-conditioned room at a temperature of 25±1[1]C and relative humidity of 55±5%, with an 11:13 h light: dark cycle. Male rats were divided into five groups of eight each and were fed the basal diet (control) or the basal diet containing the test chemical at levels of 0.06–0.25% for 2 months. Body weight and food intake were occasionally measured. Clinical signs of toxicity were daily recorded. At termination of administration, rats were killed and the blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed. Spleen weight was also measured in the rat 0.25% study. Testis was fixed with formalin, sectioned, and stained routinely with hematoxylin and eosin, and observed microscopically. Testicular sperm content and daily sperm production (DSP) were determined. One whole testis frozen at-80˚C from each rat or mouse was reweighed and homogenized in 0.9% NaCl–Triton X-100 solution. After staining by Trypan blue, unbroken nuclei of elongated spermatids were counted on the hemocytemeter. DSP and its efficiency (DSP/g testis) were determined by the division of the number of spermatids per testis, and spermatids per g testis with 6.1 for rats and 4.8 for mice. The test chemical induced toxicity in the testes. Animals feed with 0.06% test substance showed a decreased relative testicular and epidididymal weights and histopathological changes such as vaculoation of Sertoli cells, disappearance of basement membrane and degeneration of spermatids. Moreover, the daily sperm production (DSP) was significant decreased in treated animals. In contrast, the serum testosterone levels were not significant changed in treated animals compared to control. No estrogenic activity of the test substance was noted in an in vitro ER-binding assay. Therefore, LOAEL was considered to be 0.06% per day when male F344/Du Crj (Fischer) rats were treated with test substance on daily basis in feed for 2 months.

Study 4:

In a two month feeding study, the test chemical was administered in with maleCRj: CD-1 (ICR) mice. The mice were housed individually in chip-bedded plastic and stainless steel suspended cages, respectively, in an air-conditioned room at a temperature of 25± 1 ˚C and relative humidity of 55 ± 5%, with an 11:13 h light: dark cycle. Male mice were divided into five groups of eight each and were fed the basal diet (control) or the basal diet containing the test chemical at levels of 0.06–0.25% for 2 months. Body weight and food intake were occasionally measured. Clinical signs of toxicity were daily recorded. At termination of administration, rats were killed and the blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed. Spleen weight was also measured in the rat 0.25% study. Testis was fixed with formalin, sectioned, and stained routinely with hematoxylin and eosin, and observed microscopically. Testicular sperm content and daily sperm production (DSP) were determined. One whole testis frozen at-80˚C from each mice was reweighed and homogenized in 0.9% NaCl–Triton X-100 solution. After staining by Trypan blue, unbroken nuclei of elongated spermatids were counted on the hemocytemeter. DSP and its efficiency (DSP/g testis) were determined by the division of the number of spermatids per testis, and spermatids per g testis with 6.1 for rats and 4.8 for mice. The test chemical induced no significant toxic effects in absolute organ weights such as testes, accessory organ weights, liver and kidney. However, animals feed with the test substance showed histopathological changes in giant cells and Leydig cell vacuolization, while no estrogenic activity of the test substance was noted in an in vitro ER Alpha-binding assay. Therefore based on all the observations and results, LOAEL was considered to be 0.25% per day when male CRrj: CD-1 (ICR) mice were treated with the test chemical on daily basis in feed for 2 months.

Study 5:

In a chronic feeding study, the effects of 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol were investigated in male and female Wistar rats. The animals were treated with 0, 0.01, 0.03 or 0.1% per day of test substance mixed in feed. A suppression of the body weight gain was noted at 0.1% per day, and an increase in the relative liver weight was found at 0.03 and 0.1% per day in males and at 0.1% per day in females. In males, a decrease of the absolute and relative testes weights and atrophy of testicular tubules were observed at 0.03 and 0.1% per day. In addition, a spermatogenic arrest and epididymis hypospermia were noted at 0.1% per day in males. Therefore, NOAEL was considered to be 0.01% per day for males and 0.03% per day for females, while LOAEL for the males was considered to be 0.03% per day when Wistar rats were exposed to 2,2'-methylenebis(4-methyl-6-tert-butylphenol) by oral route for 18 months. The NOAELs are 4.2 mg/kg/day (100 ppm) for male and 15.1 mg/kg/day (300 ppm) for female.LOAEL for females was assessed to be 15.1 mg/kg/day(300 ppm).

Study 6:

A subchronic feeding study was performed to assess the effect of the test chemical on the reproductive parameters of the rats. The effects of the test chemical were investigated in male and female Wistar rats. The animals were treated with 0, 0.12, 0.6 or 3.0% per day of test substance mixed in feed.

Clinical signs were recorded daily. Body weight and food consumption were weekly. Hematological and serum biochemical examinations were conducted after 16 hrs starvation at week 4 and 12. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg). Urinalysis was also conducted in fresh urine for pH, protein, glucose, ketone bodies, occult blood, bilirubin and urobilin in the morning at weeks 4 and 12. At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examination. All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data. Mortality was observed in males of 0.6% and 3.0% dosed group and females of 3.0% dose group from weeks 3 to 4. Haemorrahage from the nasal cavity was observed from all the dead animals. Diarrhea was observed in two of the four of 3.0% of the males and one in four females from 3.0% dose group of dead animals. In both the sexes, severe suppression was observed in 0.6 and 3.0 % dose groups. Decreased mean food intake was observed in males of 0.6 and 3.0% dose group and females of 3.0% dose group. Significant decrease in the RBC was observed in 0.6% males at week 12, but not in treated females. Significant decrease in HGB at 3.0% dose group males in week 4, and 0.6% dose group males and all treated females at week 12. Although, not significant, a decrease in PLT in both the sexes of 0.3% were observed in week 12. Significant decrease in GLU was observed in 3.0% dose groups males and 0.6 and 3.0% dose group females at week 12. PL was significantly increased in 0.6 and 3.0% males and 0.6% females at week 4 and 0.6% and 3.0% dose group females at week 12. T-CHO and F-CHO were significantly elevated elevated in both the sexes at 0.6 and 3.0% dose groups in week 4 and thereafer. CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% at week 4 and in 0.6% and 3.0%  females at week 12. Gamma-GTP was significantly increased in 0.6 and 3.0% females at week 4 and 0.6% males and 0.6 and 3.0% females at week 12. Relative dose-dependent increase in the liver weights in males and dose-dependent increase in the absolute liver weights in females was observed. Absolute and relative testis weights were decreased in dose and time dependent manner in all treated males. In females, absolute and relative decrease in ovary weights was observed in females of 3.0% dose groups at wek 4 and 12. In male rats, testicular atrophy and appearance of the giant cells were observed in the 0.6 and 3.0% dose groups in week 4. At week 12, testicular atrophy was observed and pronounced at all the dose groups and all the male treated animals. Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed. In females, atrophy of the ovaries and uterus were apparent in the 0.6% and 3.0% groups. In both the sexes, thymus atrophy and bone marrow hypoplasia were noted  in the 0.6 and 3.0% groups. No histopathological changes were observeds in any other organs. Thus, based on all the results and observations, it was concluded that, the NOAEL was 104mg/kg/day (1200 ppm) for male and  female.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The data is from a J-check report and from a Klimisch 2 database.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive Toxicity Study:

Study 2:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Study 3:

In a two month feeding study with male F344/Crj (Fischer) rats, the test chemical was administer in F344/Crj (Fischer) rats. The rats were housed individually in chip-bedded plastic and stainless steel suspended cages, respectively, in an air-conditioned room at a temperature of 25±1[1]C and relative humidity of 55±5%, with an 11:13 h light: dark cycle. Male rats were divided into five groups of eight each and were fed the basal diet (control) or the basal diet containing the test chemical at levels of 0.06–0.25% for 2 months. Body weight and food intake were occasionally measured. Clinical signs of toxicity were daily recorded. At termination of administration, rats were killed and the blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed. Spleen weight was also measured in the rat 0.25% study. Testis was fixed with formalin, sectioned, and stained routinely with hematoxylin and eosin, and observed microscopically. Testicular sperm content and daily sperm production (DSP) were determined. One whole testis frozen at-80˚C from each rat or mouse was reweighed and homogenized in 0.9% NaCl–Triton X-100 solution. After staining by Trypan blue, unbroken nuclei of elongated spermatids were counted on the hemocytemeter. DSP and its efficiency (DSP/g testis) were determined by the division of the number of spermatids per testis, and spermatids per g testis with 6.1 for rats and 4.8 for mice. The test chemical induced toxicity in the testes. Animals feed with 0.06% test substance showed a decreased relative testicular and epidididymal weights and histopathological changes such as vaculoation of Sertoli cells, disappearance of basement membrane and degeneration of spermatids. Moreover, the daily sperm production (DSP) was significant decreased in treated animals. In contrast, the serum testosterone levels were not significant changed in treated animals compared to control. No estrogenic activity of the test substance was noted in an in vitro ER-binding assay. Therefore, LOAEL was considered to be 0.06% per day when male F344/Du Crj (Fischer) rats were treated with test substance on daily basis in feed for 2 months.

Study 4:

In a two month feeding study, the test chemical was administered in with maleCRj: CD-1 (ICR) mice. The mice were housed individually in chip-bedded plastic and stainless steel suspended cages, respectively, in an air-conditioned room at a temperature of 25± 1 ˚C and relative humidity of 55 ± 5%, with an 11:13 h light: dark cycle. Male mice were divided into five groups of eight each and were fed the basal diet (control) or the basal diet containing the test chemical at levels of 0.06–0.25% for 2 months. Body weight and food intake were occasionally measured. Clinical signs of toxicity were daily recorded. At termination of administration, rats were killed and the blood was collected. Preputial glands, testes, epididymides, prostate glands, seminal vesicles with coagulation glands, kidneys, and liver were dissected out and weighed. Spleen weight was also measured in the rat 0.25% study. Testis was fixed with formalin, sectioned, and stained routinely with hematoxylin and eosin, and observed microscopically. Testicular sperm content and daily sperm production (DSP) were determined. One whole testis frozen at-80˚C from each mice was reweighed and homogenized in 0.9% NaCl–Triton X-100 solution. After staining by Trypan blue, unbroken nuclei of elongated spermatids were counted on the hemocytemeter. DSP and its efficiency (DSP/g testis) were determined by the division of the number of spermatids per testis, and spermatids per g testis with 6.1 for rats and 4.8 for mice. The test chemical induced no significant toxic effects in absolute organ weights such as testes, accessory organ weights, liver and kidney. However, animals feed with the test substance showed histopathological changes in giant cells and Leydig cell vacuolization, while no estrogenic activity of the test substance was noted in an in vitro ER Alpha-binding assay. Therefore based on all the observations and results, LOAEL was considered to be 0.25% per day when male CRrj: CD-1 (ICR) mice were treated with the test chemical on daily basis in feed for 2 months.

Study 5:

In a chronic feeding study, the effects of 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol were investigated in male and female Wistar rats. The animals were treated with 0, 0.01, 0.03 or 0.1% per day of test substance mixed in feed. A suppression of the body weight gain was noted at 0.1% per day, and an increase in the relative liver weight was found at 0.03 and 0.1% per day in males and at 0.1% per day in females. In males, a decrease of the absolute and relative testes weights and atrophy of testicular tubules were observed at 0.03 and 0.1% per day. In addition, a spermatogenic arrest and epididymis hypospermia were noted at 0.1% per day in males. Therefore, NOAEL was considered to be 0.01% per day for males and 0.03% per day for females, while LOAEL for the males was considered to be 0.03% per day when Wistar rats were exposed to 2,2'-methylenebis(4-methyl-6-tert-butylphenol) by oral route for 18 months.The NOAELs are 4.2 mg/kg/day (100 ppm) for male and 15.1 mg/kg/day (300 ppm) for female.LOAEL for females was assessed to be 15.1 mg/kg/day(300 ppm).

Study 6:

A subchronic feeding study was performed to assess the effect of the test chemical on the reproductive parameters of the rats. The effects of the test chemical were investigated in male and female Wistar rats. The animals were treated with 0, 0.12, 0.6 or 3.0% per day of test substance mixed in feed.

Clinical signs were recorded daily. Body weight and food consumption were weekly. Hematological and serum biochemical examinations were conducted after 16 hrs starvation at week 4 and 12. Hematological parameters examined were Red blood cells (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), mean hemoglobin concentration (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), platelets (PLT) and white blood cells (WBC). Serum biochemical examinations included total protein (T-PRO), albumin (ALB), blood urea nitrogen (BUN), creatinine (CRN), uric acid (UA), glucose (GLU), non-esterified fatty acids (NEFA), phospholipids (PL), triglycerides (T-GLY), total cholesterol (T-CHO), free cholesterol (F-CHO), alkaline phosphatase (ALP), amylase (AMY), cholinesterase (CHE), aspartate aminotransferase (AST), alanine aminotransferase (ALT), 7-glutamyl transpeptidase ( 7 -GTP) , 2-hydroxybutyrate dehydrogenase (HBDH), leucine aminopeptidase (LAP) and lactate dehydrogenase (LDH) and calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), chloride (CI) and magnesium (Mg). Urinalysis was also conducted in fresh urine for pH, protein, glucose, ketone bodies, occult blood, bilirubin and urobilin in the morning at weeks 4 and 12. At autopsy, the weights of the brain, heart, lungs, liver, kidneys, spleen, adrenals, testes, ovaries, pituitary and thyroid glands were measured. The above-mentioned organs and the salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiatic nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle and bone marrow (femur and sternum) were fixed in 10% buffered formalin solution for routine histological processing and examination. All quantitative data except for the histopathological findings were statistically analyzed by one-way analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's multiple comparison procedures. Histopathological data were statistically analyzed with the cumulative Chi-squares test which is known to be useful for analysis of categorical data. Mortality was observed in males of 0.6% and 3.0% dosed group and females of 3.0% dose group from weeks 3 to 4. Haemorrahage from the nasal cavity was observed from all the dead animals. Diarrhea was observed in two of the four of 3.0% of the males and one in four females from 3.0% dose group of dead animals. In both the sexes, severe suppression was observed in 0.6 and 3.0 % dose groups. Decreased mean food intake was observed in males of 0.6 and 3.0% dose group and females of 3.0% dose group. Significant decrease in the RBC was observed in 0.6% males at week 12, but not in treated females. Significant decrease in HGB at 3.0% dose group males in week 4, and 0.6% dose group males and all treated females at week 12. Although, not significant, a decrease in PLT in both the sexes of 0.3% were observed in week 12. Significant decrease in GLU was observed in 3.0% dose groups males and 0.6 and 3.0% dose group females at week 12. PL was significantly increased in 0.6 and 3.0% males and 0.6% females at week 4 and 0.6% and 3.0% dose group females at week 12. T-CHO and F-CHO were significantly elevated elevated in both the sexes at 0.6 and 3.0% dose groups in week 4 and thereafer. CHE activity was significantly decreased in both males and females receiving 0.6 or 3.0% at week 4 and in 0.6% and 3.0%  females at week 12. Gamma-GTP was significantly increased in 0.6 and 3.0% females at week 4 and 0.6% males and 0.6 and 3.0% females at week 12. Relative dose-dependent increase in the liver weights in males and dose-dependent increase in the absolute liver weights in females was observed. Absolute and relative testis weights were decreased in dose and time dependent manner in all treated males. In females, absolute and relative decrease in ovary weights was observed in females of 3.0% dose groups at wek 4 and 12. In male rats, testicular atrophy and appearance of the giant cells were observed in the 0.6 and 3.0% dose groups in week 4. At week 12, testicular atrophy was observed and pronounced at all the dose groups and all the male treated animals. Decreased amount of spermatogenesis was observed in all treated males in week 4 and thereafter. Interstitial edema was also observed in testes of the male rats at all dose groups at week 12. At week 4 and 12, epididymis atrophy and hypospermia at 3.0% males and atrophy of the seminal vesicles and prostate in the 0.6% and 3.0% groups were observed. In females, atrophy of the ovaries and uterus were apparent in the 0.6% and 3.0% groups. In both the sexes, thymus atrophy and bone marrow hypoplasia were noted  in the 0.6 and 3.0% groups. No histopathological changes were observeds in any other organs. Thus, based on all the results and observations, it was concluded that, the NOAEL was 104mg/kg/day (1200 ppm) for male and  female.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Weight of evidence based on the information of data of test chemical.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 421 (Reproductive and Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The above test substance is referred to as the test chemical in the study.
GLP compliance:
not specified
Specific details on test material used for the study:
- Molecular weight (if other than submission substance): 256.343 g/mol
- Substance type: Organic
- Physical state: White Powder
- Impurities (identity and concentrations): No Data Available
Species:
other: Study 2, 3 and 4: Rat
Strain:
other: Study 2: Crj: CD (SD) IGS, (SPF); Study 3: Sprague-Dawley; Study 4: Walter Reed Carworth Farms
Details on test animals or test system and environmental conditions:
Study 2: TEST ANIMALS
- Source: Charles River Japan
- Age at study initiation: 10 week old
- Weight at study initiation: 332-383 g for male, 206-238 g for female.
- Fasting period before study: Not available
- Housing: Stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.
- Diet (e.g. ad libitum): The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.)
- Water (e.g. ad libitum): The drinking water was freely taken in all of the tap water
- Acclimation period: Not available

ENVIRONMENTAL CONDITIONS .
- Temperature (°C): 20 to 26 ° C.TEST ANIMALS
- Source: No data available
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To:

Study 4: TEST ANIMALS
- Source: No data available
- Age at study initiation: No data available
- Weight at study initiation:200 g
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%): No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To:
Route of administration:
other: Study 2, 3: oral: gavage; Study 4: Oral: Feed
Vehicle:
other: Study 2: 5% gum arabic solution; Study 3: Mazola corn oil; Study 4: Diet
Details on exposure:
Study 2: Details on exposure
PREPARATION OF DOSING SOLUTIONS: The test substance was prepared by suspending it in a 5% gum arabic solution. The test substance was converted to purity, and the dose was expressed in terms of the original weight. Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation. Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance.

DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available

VEHICLE
- Justification for use and choice of vehicle (if other than water): No Data Available
- Concentration in vehicle: Concentration of 5, 20 and 80 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): No Data Available
- Lot/batch no. (if required): No Data Available
- Purity: No Data Available

Study 3: PREPARATION OF DOSING SOLUTIONS: Prior to the start of the study, homogeneity of TMP in corn oil, storage stability, and stability under conditions simulating the dosing procedures were conducted at concentrations of 2 and 40 mg/ml TMP in corn oil. TMP was shown to be stable in vehicle for at least 35 days under ambient and refrigerated conditions.

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil
- Concentration in vehicle: 0, 10, 100 or 200 mg/kg/day
- Amount of vehicle (if gavage): 5 ml/kg/day
- Lot/batch no. (if required): No data available
- Purity: No data available

Study 4: PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test chemical was mixed with the diet before administration in animals.
- Concentration in vehicle: 0 or 0.25 g
- Amount of vehicle (if gavage): Not available
- Lot/batch no. (if required): Not available
- Purity: Not available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No Data Available
Details on mating procedure:
Study 2: - M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy vaginal plug
- After … days of unsuccessful pairing replacement of first male by another male with proven fertility. Not available
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not available
- After successful mating each pregnant female was caged (how):Not available
- Any other deviations from standard protocol:Not available

Study 3: F0 Males = 4 weeks (2 weeks pre-breeding, 2 weeks mating)
Females = 10 weeks (2 weeks pre-breeding, 2, weeks mating, 3 weeks gestation and 3 weeks lactation)
F1Offspring = 7 weeks post-weaning

Study 4: The females were mated with male rats and positively mated animals were used in the study.
Duration of treatment / exposure:
Study 2: Male: 50-52 days
Female: 40 to 48 days.

Study 3: No Data Available

Study 4: Females were treated for 22 days.
Frequency of treatment:
Study 2, 3 and 4: Daily
Duration of test:
Study 2: Approximately 68 days
Study 3: F1 Generation dosed until ~ PND 80
Study 4: No data available
Remarks:
Study 2: Doses / Concentrations:
12.5, 50, 200, 800 mg/kg/day (in 5% gum arabic)
Basis:
no data
Study 3: Doses / Concentrations:
0, 10, 100, and 200 mg/kg/day
Basis:
no data
Study 4: Doses / Concentrations:
0 or 0.25 g/day (250 mg/day)
Basis:
nominal in diet
No. of animals per sex per dose:
Study 2: Control-12 female and 12 male
12.5 mg/kg/day- 12 female and 12 male
50 mg/kg/day-12 female and 12 male
200 mg/kg/day-12 female and 12 male
800 mg/kg/day-12 female and 12 male

Study 3: Treated animals
Control: 10 males, 10 females
10 mg/kg/day: 10 males, 10 females
100 mg/kg/day: 10 males, 10 females
200 mg/kg/day: 10 males, 10 females

Treated recovery animals
Control: 5 males, 5 females
200 mg/kg/day: 5 males, 5 females

Non-pregnated animals (“28-days females”)
Control: 5 females
200 mg/kg/day: 5 females

Study 4: No Data Available
Control animals:
yes, concurrent vehicle
Details on study design:
Study 2: No Data Available
Study 3: - Dose selection rationale: Doses were selected for this study based on the results of a 10-day dose range-finding (RF) study. In this study, TMP was dosed by gavage to male and female rats for 10 consecutive days at 0, 100, 300 or 1000 mg/kg/day. Body weight loss over the 10-day period was observed for males at 1000 mg/kg/day and females at 300 and 1000 mg/kg/day. In addition, weight gain in males at 300 mg/kg/day was reduced by 43% compared to the controls. Therefore, 300 mg/kg/day was considered to toxic for the OECD 422 study design.
- Rationale for animal assignment (if not random):No data available
- Other: No other details available
Study 4: - Dose selection rationale: No data available

- Rationale for animal assignment (if not random): No data available

- Other: Walter Reed-Carworth Farms strain rats in their first gestation were used. After positive mating the females were randomly distributed into experimental and control groups and the former were given per os or directly into diet the test compound. Twenty-two days after positive mating the pregnant rat was sacrificed in 100% nitrogen and after 21 minutes the young delivered by caesarian section. Both horns of the uterus were completely incised and a careful macroscopic survey made for resorption sites.

Maternal examinations:
Study 2: (1) General condition: The general condition and the presence or absence of death were observed twice a day before and after administration.
(2) Sex cycle: The sex cycle was observed once a day from the administration start date to the mating confirmation date. In addition, when the estrus period was observed over 2 consecutive days, it was counted as 1 time.
(3) Weight measurement: Body weights were measured twice a week during the 14 days before the mating and during the mating period, at 0, 7, 14 and 21 gestation during gestation, on 0 and 4 days of feeding during the feeding period, respectively .
(4) Food consumption measurement: Food consumption was measured twice a week until 14 days before the start of the mating. Also, during pregnancy, gestation was measured on 2, 9, 16 and 21 gestation, and 4 days after nursing during gestation period.
(5) Observation of delivery status: Mating females were allowed to spontaneously deliver, and the presence or absence of abnormality in labor condition and confirmation of end of delivery were confirmed once a day from day 21 of pregnancy until 25th day of pregnancy. If delivery was completed at 10:00 am, that day was taken as nursing day 0.
(6) Animals not delivered by 25th day gestation: Females who did not deliver until 25th day of pregnancy were necropsied after lethality from the abdominal aorta under ether anesthesia.
(7) Observation of nursing condition and autopsy: Maternal animals were observed for nursing condition once a day until 4th day of nursing and autopsied after death from the abdominal aorta under ether anesthesia on the day when all newborn cases died or on nursing 4th day, necropsied, and the number of implantation traces and corpus luteum were conted. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight.
(8) Histopathology examination: For the ovary, a paraffin-embedded specimen was prepared according to an ordinary method. HE stained tissue specimens were prepared for the control group and the ovaries of the 800 mg / kg group and histopathological examination was performed.
3) Effects of parent animals on reproductive development: Male and female rats administered for 14 days were mated together in a 1: 1 combination within the same group. The mating period was limited to 14 days, but all cases mated in 8 days. In addition, we continued cross-living until we confirmed copulation.
Mating was confirmed approximately every morning at a fixed time and a female who confirmed sperm or vaginal plug in vaginal plaque was a mating animal and the day was counted as the 0th day of pregnancy.

Study 3: CAGE SIDE OBSERVATIONS: Yes
Time schedule: Performed on all initial animals once during quarantine and at least once per week for F0 animals during pre-breed, mating, gestation and lactation treatment periods, and on 5 F1 females and 5 F1 males once midway during the post-wean exposure period.

CLINICAL OBSERVATIONS: Yes
Time schedule: At least once daily for F0 females and F1 offspring.

BODY WEIGHT: Yes
Time schedule for examinations:
- Body weights for F0 male and female rats were recorded weekly during the pre-breed period.
- F0 females were weighed weekly during gestation and lactation.
- Selected F1 offspring were weighed weekly weaning through scheduled sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: All 28-day females, for 5 randomly selected parental F0 males and females per dose group, and for 5 F1 adult males and females per dose group.
- Parameters examined: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, RBC indices and prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: No data available
- How many animals: All 28-day females, for 5 randomly selected parental F0 males and females per dose group, and for 5 F1 adult males and females per dose group.
- Parameters examined: Sodium, potassium, chloride, glucose, total cholesterol, blood urea nitrogen (BUN), creatinine, total protein and albumin, and 2 enzymes indicative of hepatocellular effects (alanine aminotransferase and aspartate aminotransferase).

URINALYSIS: Yes
- Time schedule for collection of urine: At necropsy
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters examined: Specific gravity and pH

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Performed on F0 males prior to dosing during quarantine and weekly to termination at sd 28.
Performed on F0 females prior to dosing and weekly during pre-breed, mating, gestation, and lactation and on recovery animals (male and female) prior to dosing, weekly during dosing, and once during the recovery period.
- Dose groups that were examined: 0, 10, 100 and 200 mg/kg/day
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Five F0 males and 5 F0 females per dose group were evaluated for auditory function, motor activity, and assessment of grip strength prior to necropsy.

POST-MORTEM EXAMINATIONS: Yes
All F0 parental animals, non-selected F1 weanlings, and retained F1 adults were necropsied with complete histologic evaluation of the 28-day females and for 5 selected F0 and F1 males and females in the 0 and 200 mg/kg/day groups.

Histopathology was performed on control and high-dose animals only.
Organs examined: Brain, thymus, heart, liver, spleen, kidneys, adrenal glands, testes, epididymides, prostate, uterus with vagina and cervix and paired Ovaries.

Study 4: The number and distribution of resorption, the number of implantations, litters, normal fetuses and litters with resorptions were observed.
Ovaries and uterine content:
Study 2: In uterine content, the number of implantation traces and corpus luteum were conted. The ovaries were weighed and fixed in 20% neutral buffered formalin. The relative weight was also calculated by dividing the ovarian weight by the final body weight.

Study 3: The uterine examined after termination: Yes
Examinations included:
Organ weight: Yes
Number of live fetuses: Yes
Number of dead fetuses: Yes
Number of corpora lutea: No
Number of implantations : Yes
Number of resorptions: No

Study 4: Horns of the uterus were completely incised and a careful macroscopic survey made for resorption sites

Fetal examinations:
Study 2: (1) Observation at birth: At birth the number of total births and sex, the number of stillborn babies, the number of neonates and the presence or absence of outer table abnormalities were observed.
(2) Observation of newborns: Neonates were observed for general condition and the presence or absence of death once a day.
(3) Body weight: Body weight was measured on day 0 of nursing (birthday) and 4th day.
(4) Autopsy: The surviving child was sacrificed by abdominal aorta from the abdominal aorta under necropsy under ether anesthesia on 4th day of nursing and then necropsied.

Study 3: Fetal weight: Yes
Litter size: Yes
Sex: Yes
Survival index: Yes
External anomalies: Yes
Soft tissue anomalies: Yes
Skeletal anomalies: Yes
Postnatal mortality: Yes

Study 4: No Data Available
Statistics:
Study 2: For statistical analysis, homodispersity was tested by the Bartlett method. In the case of equi-variance, variance analysis was performed by one-way method, and if significant, it was performed by the Dunnett method. On the other hand, when it was not recognized as equal variance, we performed analysis by the one-way method using rank order (Kruskal-Wallis test), and if significant, we used Dunnett type test method using ranking. Mating rate, conception rate and birth rate were determined by χ ^ 2 test. In histopathological examination, the toxicological effects were suggested in the 800 mg / kg group, and findings of organs and tissues examined for other groups were ranked using comparison between groups with the control group Dunnett type test method was used. Therefore, when a significant difference was observed with the control group, the dose reactivity test was conducted using Cochran · Armitage trend test.

Study 3: For all statistical tests, p<0.05 (one- or two-tailed) was used as the criterion for significance.
Indices:
Study 2: Resorption and Implantation Index, Pup Viability index, Gestation Index

Study 3: F0
- Females: Mating, fertility, gestational, percentage postimplantation loss per litter
- Males: Mating, fertility and pregnancy.

F1
Stillbirth index, live birth index, survival index, lactation index and sex ratio.

Study 4: No data available
Historical control data:
Study 2: No Data Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Study 2: Transient salivation was observed in the 200 and 800 mg / kg group in general condition observation. In addition, loose stools were observed in each group including the control group, but no relation with the dose was observed.
Study 3: Clinical signs of rooting post-dosing were obseClinical signs of rooting post-dosing were observed in a dose-related manner throughout the study. These signs are considered to be a response to taste aversion to the dosing solution and not an indication of toxicity, per se.rved in a dose-related manner throughout the study. These signs are considered to be a response to taste aversion to the dosing solution and not an indication of toxicity, per se.
Study 4: no effects observed
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Study 2: Death and moribund cases were not observed in either group.
Study 3: No Data Available
Study 4: no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 2: Before the mating, there was no significant difference in body weight at any measurement day in each administration group compared with the control group.
During the gestation period, there was no significant difference in body weight at any measurement day in the 12.5, 50 and 200 mg / kg group compared with the control group. In the 800 mg / kg group, there was a significant lower value of body weight on 0 to 21 days of pregnancy than in the control group.
period, there was no significant difference in body weight at any measurement day in the 12.5 and 50 mg / kg group compared to the control group. In the 200 and 800 mg / kg group, a significant lower value of body weight was seen on 4th day of nursing compared with the control group.

Study 3:No treatment-related effects on body weight or food consumption for the adult animals in the study.

Study 4: no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Study 2: Before the mating, there was no significant difference in food consumption on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 mg / kg group, a significant lower value of food intake was observed on days 3 and 6 compared to the control group. In the 800 mg / kg group, a significant low value of food intake was seen on the 3rd day of administration compared with the control group. During the gestation period, there was no significant difference in food intake on any measurement day in the 12.5 and 50 mg / kg group compared with the control group. In the 200 and 800 mg / kg group, a significant lower value of food intake was seen on the 2nd day of pregnancy than in the control group. There was no significant difference in food intake in the 12.5 and 50 mg / kg group during the nursing period compared with the control group. In the 200 and 800 mg / kg group, a significant low value of food intake was seen on the 4th day of nursing compared with the control group.

Study 3:No treatment-related effects on body weight or food consumption for the adult animals in the study.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Study 3: no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Study 3: no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
Study 3: no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Study 3: There were no significant differences for any parameters included in the Functional observational battery, including home-cage observations, handling observations, sensory and neuromuscular observations or open field observations. There were no differences among groups for any parameters evaluated for auditory startle, motor activity or grip strength.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Study 2: There was no significant difference in body weight at day of necropsy compared with control group in 12.5 and 50 mg / kg group. In the 200 and 800 mg / kg group, there was a significant lower value of body weight on the necropsy day compared to the control group. There was no significant difference in the absolute and relative weights of ovaries in each treatment group compared to the control group.

Study 3: no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Study 2: In the control group, renal pelvic dilation was found in one case. No abnormality was found in any of the administration groups.

Study 3: No Data Available
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Study 3: There were no changes in histopathology related to treatment.
Other effects:
not specified
Details on results:
No Data Available
Number of abortions:
not specified
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 and 50 mg / kg groups, there was no significant difference in number of corpus luteum, number of implantation traces and landing rate compared to the control group. In the 200 mg / kg group, although there was no significant difference compared with the control group, there was a tendency for the number of corpus luteum and the number of implantation traces to be low. In the 800 mg / kg group, the number of corpus luteum and the number of implantation traces were significantly lower than in the control group.

Study 3:No Data Available
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Study 3: no effects observed
Study 4: Litters with resorptions were found to be 37.5%.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Study 3: no effects observed
Study 4: Litters with resorptions were found to be 37.5%.
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed. There was no abnormality in each group in the observation of the external table of the newborn. There was no abnormality in each group in the general condition of the newborn.

Study 3: Live birth and stillbirth indices were unaffected, as were the survival indices for PND 0-4, 4-7, 7-14 and 14-21 as well as the lactational index for PND 4 (postcull) through 21.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical biochemistry
clinical signs
dead fetuses
effects on pregnancy duration
food consumption and compound intake
gross pathology
haematology
mortality
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
urinalysis
Remarks on result:
other:
Remarks:
Not Specified
Abnormalities:
not specified
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 and 50 mg / kg group, there was no significant difference in body weights of sexes on 0 and 4 days of nursing compared with the control group. In the 200 mg / kg group, there was a significant high value of sex and body weight on 0th day of nursing compared to the control group, but because there was no significant difference in the 800 mg / kg group, it was not due to administration It is judged. In the 800 mg / kg group, there was no significant difference in male body weight on 4th day of nursing compared to the control group, but there was no significant difference in female body weight on 4th day of nursing though there was no significant difference.

Study 3: There were no effects on F1 male or female body weights at sacrifice on PND 21 at any dose.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Study 2: In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed.

Study 3: No Data Available
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
Study 2 and 3: no effects observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
Study 3: no effects observed
Visceral malformations:
no effects observed
Description (incidence and severity):
Study 2 and 3: no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
No data Available
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Developmental effects observed:
not specified
Treatment related:
not specified
Conclusions:
Study 2: In the 12.5 and 50 mg / kg groups, there was no significant difference in the number of surviving children and survival rate on 4th day of nursing compared to the control group. In the 200 mg / kg group, there was a significant low value of the number of surviving children on 4th day of nursing compared to the control group. In the 800 mg / kg group, although there was no significant difference compared with the control group, a low value trend of the number of surviving children on nursing 4th day was observed.

Study 3: NOAEL was considered to be ≥200 mg/kg/day in both the F0 and the F1 generation in CD (Sprague-Dawley) rats exposed to the test chemical.

Study 4: The LOAEL was considered to be 250 mg/day in pregnant female mice when exposed to the test chemical.
Executive summary:

Developmental Toxicity Study:

The Data from developmental toxicity studies are as follows:

Developmental Toxicity Study 2:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Developmental Toxicity Study 3:

In a reproductive toxicity study, the toxic effect of the test chemical was evaluated in male and female CD (Sprague-Dawley) rats. The test chemical was administered by gavage once daily at 0, 10, 100 or 200 mg/kg/day to parental rats through pre-breed, mating, gestation and lactation, and by direct dosing to F1 offspring from weaning to scheduled sacrifice. Treatment resulted in no adult F0 parental toxicity (for pregnant females or non-pregnant females) and there was no evidence of toxicity (systemic, reproductive, or developmental) in F1 offspring animals. In addition, there were no treatment or dose-related findings in any of the systemic, reproductive, developmental, or neurobehavioral endpoints evaluated in-life, at necropsy, in gross or microscopic pathology, male andrology, clinical chemistry, hematology or urinalysis. Therefore, NOAEL was considered to be ≥200 mg/kg/day in male and female of the parental generation as well as for the F1 generation when CD (Sprague-Dawley) rats were exposed to the test chemical on a daily basis.

Developmental Toxicity Study 4:

In present study, the Walter Reed Carworth Farms strain rat was studied after being orally treated with 250 mg of the test chemical. The test chemical was administered through the diet. The weight of rats were 200 g at initiation. Fetal resorptions for the rats were studied. No clinical signs, mortality, body weights and effects on feed consumptions were observed. Marginal differences was noted in resorption of treated rats compared to control. The percentage of resorption was reported to be 37.5%. Therefore. LOAEL was considered to be 250 mg/day when pregnant female Walter Reed Carworth Farms strain rat were treated with the test chemical by oral route for 22 days.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The data is from a J-check report and from a Klimisch 2 database.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental Toxicity Study:

The Data from developmental toxicity studies are as follows:

Developmental Toxicity Study 2:

A combined repeated and reproductive/developmental toxicity test by oral administration to rats of the test chemical was conducted to investigate the effects on the reproductive ability of the male and foster parents and development and development of the next generation .The dose was 800 mg / kg as the highest dose, and 200, 50 and 12.5 mg / kg as below.As a control, a vehicle (5% aqueous gum arabic solution) administration group was provided. The test substance was prepared by suspending it in a 5% gum arabic solution.The test substance was converted to purity, and the dose was expressed in terms of the original weight.Since it was confirmed that the prepared solution had no problem of stability even after storage for 7 days under refrigeration / light-shielding conditions and further at room temperature under light-shielding conditions for 4 hours, the preparation solution of each concentration was prepared , Stored under refrigerated / light-proof conditions, and used within 7 days after preparation.Also, as a result of confirming the concentration of the test substance in the administration solution at each concentration used at the administration start date and the end date of the male administration period, there was no problem with the concentration of the test substance. 8-week-old Sprague-Dawley male and female rats [Crj: CD (SD) IGS, (SPF)] were purchased from Charles River Japan.The obtained animals were subjected to a quarantine period of 5 days and a subsequent acclimatization period of 7 days, and animals that were not abnormal in general state and body weight transition and had no abnormality by sexual cycle observation were grouped.Grouping was carried out on the administration start date so that the average body weight and variance of each group were almost equal by random sampling method after dividing body weight by stratification using a computer.The number of animals in one group was 12 male and female. The animals were kept in a room kept at room temperature 20 to 26 ° C., humidity 40 to 70%, light and dark each for 12 hours (lighting: 6 am to 6 pm), ventilation frequency 12 times / hour.During the quarantine / acclimatization period, stainless steel cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel cage.The mother animals were individually transferred to a plastic cage containing autoclaved bedding (Sunflake, Japan Charles River Co., Ltd.) on the 18th day of pregnancy to allow natural delivery and nursing.The feed was made free from solid feed (CRF-1, Oriental Yeast Industry Co., Ltd.), and the drinking water was freely taken in all of the tap water. The administration route was selected for oral administration.Upon administration, it was forcibly orally administered using a polypropylene disposable syringe fitted with a metal oral gastric tube.In the males, the volume of the liquid to be administered was calculated as 10 mL / kg based on the administration day or the weight on the measurement day closest to the administration day.In females, the body weight of the measurement day closest to the administration day or the administration day before the mating and during the mating period, the body weights of 0, 7, 14, and 21 days of pregnancy during gestation, the body weight of day 0 nursing during the nursing period As a standard, calculated at 10 mL / kg.The number of administrations was once a day. The age at the start of administration was 10 weeks for both males and females, the body weight ranged from 332 to 383 g for males and from 206 to 238 g for females. In males, no death or moribund cases were observed in any group.In general condition and body weight, no change due to administration was observed.Feeding amount was transient low in the 800 mg / kg group.Autopsy showed atrophy of testis and epididymis in 200 mg / kg group, atrophy of testis, epididymis and seminal vesicle in 800 mg / kg group.In the organ weight, low absolute and relative weights of testis and epididymis were observed in groups of 200 mg / kg or more.Sperm examination showed an increase in spermatozoa rate, survival sperm ratio, survival sperm ratio, sperm count, sperm count per gram of epididymal tail, and malformed sperm ratio in the 50 and 200 mg / kg group .In the 800 mg / kg group, no active spermatozoa was observed, and the increase tendency of malformed sperm, the number of sperm and the low number of sperm per 1 g of epididymal body tail were observed.Histopathological examination showed giant cell formation in the testes in the 50 mg / kg group, atrophy of the seminiferous tubules in the testis in the 200 mg / kg group, degeneration of the seminiferous tubules, spermic depletion and giant cell formation, spermatozoa in the epididymis In the 800 mg / kg group, atrophy of seminiferous tubules and spermatozoa in the epididymis were observed in the testes. No deaths or moribund cases were observed in females in either group.In general condition, no change due to administration was observed.Weight gain was suppressed in the nursing stage in the 200 mg / kg group, and increased in the pregnancy and nursing period in the 800 mg / kg group.Feeding levels were low in the 200 and 800 mg / kg groups before mating, during pregnancy and nursing.Necropsy, organ weight and histopathological examination showed no change due to administration. In sperm examination and histopathological examination, as described above, changes were observed in the group of 50 mg / kg or more. There was no change due to the administration in the number of estruses, copulation rate, number of days required for copulation, conception rate, gestation period, delivery status, and nursing condition.In the 800 mg / kg group, one female who was unable to deliver a newborn baby and one mother who died all the newborn at the nursing stage were observed.In addition, in the 800 mg / kg group, the low or low tendency of sex and body weight in sex and body weight was observed in the number of corpus luteum, number of implantation traces, number of total births, number of neonates on 0 and 4 nursing, fertility rate, There was a tendency for the number of births to be high.In the 200 mg / kg group, the number of corpus luteums, the number of implantation traces, the total number of babies born, and the low or low tendency of the number of newborns on 0 and 4 nursing were observed.There was no change due to the administration in the external table, general condition and necropsy of the newborn. As described above, the general toxicological ineffective amount of thed test chemical is 50 mg / kg in males and the sperm test results and testicular histopathological examination It was considered to be 50 mg/kg / day because it was found that 12.5 mg / kg / day was affected by the results and 200 mg / kg in females suppressed body weight gain and low food intake .In addition, reproductive developmental toxicological ineffective dose was 12.5 mg / kg / day in females because it affected sperm examination results and testicular histopathological examination results by 50 mg / kg administration in males, 200 A low tendency of the number of corpus luteum and the number of implantation traces was observed by mg / kg administration, so 50 mg / kg / day was administered at a dose of 50 mg / kg / day, and in children, 200 mg / kg administration resulted in a low number of newborns on day 0 and day 4 It was considered to be 50 mg / kg / day.

Developmental Toxicity Study 3:

In a reproductive toxicity study, the toxic effect of the test chemical was evaluated in male and female CD (Sprague-Dawley) rats. The test chemical was administered by gavage once daily at 0, 10, 100 or 200 mg/kg/day to parental rats through pre-breed, mating, gestation and lactation, and by direct dosing to F1 offspring from weaning to scheduled sacrifice. Treatment resulted in no adult F0 parental toxicity (for pregnant females or non-pregnant females) and there was no evidence of toxicity (systemic, reproductive, or developmental) in F1 offspring animals. In addition, there were no treatment or dose-related findings in any of the systemic, reproductive, developmental, or neurobehavioral endpoints evaluated in-life, at necropsy, in gross or microscopic pathology, male andrology, clinical chemistry, hematology or urinalysis. Therefore, NOAEL was considered to be ≥200 mg/kg/day in male and female of the parental generation as well as for the F1 generation when CD (Sprague-Dawley) rats were exposed to the test chemical on a daily basis.

Developmental Toxicity Study 4:

In present study, the Walter Reed Carworth Farms strain rat was studied after being orally treated with 250 mg of the test chemical. The test chemical was administered through the diet. The weight of rats were 200 g at initiation. Fetal resorptions for the rats were studied. No clinical signs, mortality, body weights and effects on feed consumptions were observed. Marginal differences was noted in resorption of treated rats compared to control. The percentage of resorption was reported to be 37.5%. Therefore. LOAEL was considered to be 250 mg/day when pregnant female Walter Reed Carworth Farms strain rat were treated with the test chemical by oral route for 22 days.

Justification for classification or non-classification

The test chemical does not exhibit toxicity to the reproductive system and developmental parameters within the doses mentioned in the study end points. Thus, tthe test chemical may not be classified under CLP regulation.

Additional information