Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The Reproductive Toxicity Data:

In the present study, one generation reproductive toxicity test of test chemical was carried out and the toxicity of reproductive to male and female animals of test chemical was examined. In the study, animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed. The results of the parenteral examination revealed that, no mortality in treated animals at 10, 40 and 160mg/kg/day for 10 weeks,including mating period, the clinical signs examination of animlas shows, nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed. Salivation was observed in each administration group of the test substance.No test chemical related chnages on food consumption and body weight changes were observed. Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes.  There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group. There was no change depending on the dose of the test substance in reproductive organsof male and females. The histopathological study relealed no abnormality in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group.  Unilateral seminal vesicle atrophy was observed in each group including the control group. No abnormality were seen on seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina. The fetal examination results revealed, deaths in 4 offspings of control group and 2 offsping of 10 mg / kg administration group, respectively. In addition, 3 out of 6 cases in the 40 mg / kg administration group and 22 cases out of 27 cases in the 160 mg / kg administration group were judged as stillbirth, but no clinical signs of toxicity on survived pups. There was no significant difference in body weight in male and female  between the control group and each test group administered group at any time. No gross pathological changes were observed, At necropsy of born pups, morphological changes including malformations and mutations were observed in  born pups 1-2 in each group including the control group. From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Data is from Ministry of Health, Labor and Welfare (MHW) report
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Principles of method if other than guideline:
One generation reproductive toxicity test of test chemical was carried out and the toxicity of reproductive to male and female animals of test chemical was examined
GLP compliance:
yes
Limit test:
no
Justification for study design:
No data available
Species:
rat
Strain:
other: Sprague-Dawley strain (Crj: CD) rats
Details on species / strain selection:
No data available
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Tsukuba breeding center, Charles River Japan Co., Ltd.
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: housed in a metallic wire mesh floor cage in a breeding room
- Use of restrainers for preventing ingestion (if dermal): yes/no: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): tap water, provided ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ° C
- Humidity (%): 50 to 65%
- Air changes (per hr): about 15 times / hour
- Photoperiod (hrs dark / hrs light): lighting for 12 hours
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Remarks on MMAD:
No data available
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution was prepared by mixing the test chemical in vehicle with a magnetic stirrer

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: No data available
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): No data available
- Purity:No data available
Details on mating procedure:
Matings were conducted in males and females 1: 1 within the same group for a period of 3 weeks, and confirming the presence of sperm in the vaginal smear and confirming the vaginal plug every morning.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Duration of treatment / exposure:
10 weeks (71 days of administration)
Frequency of treatment:
Daily
Details on study schedule:
No data available
Remarks:
0 (vehicle control)= 25male and 25female
10mg/kg=25male and 25female
40mg/kg=25male and 25female
160 mg/kg=25male and 25female
No. of animals per sex per dose:
0 (vehicle control)= 25male and 25female
10mg/kg=25male and 25female
40mg/kg=25male and 25female
160 mg/kg=25male and 25female
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed.
Positive control:
No data available
Parental animals: Observations and examinations:
Parent animals were observed for mortality, general condition, change in weight, food intake, estrus cycle assessment.
Oestrous cyclicity (parental animals):
Estrus cycle were determined before 2 weeks of start of mating confirmation day, two weeks before the start of administration and two weeks after the start of the administration.
Sperm parameters (parental animals):
Spermatogenesis cycle was observed in males
Litter observations:
Number of births, litter count and Weight measurement
Postmortem examinations (parental animals):
Animals were sacrificed to determined presence or absence of abnormalities of major organs thoracic abdomen, including the pituitary glands, stomach, testis, epididymis, coagulated glands, seminal vesicles, and prostate in males and pituitary, stomach, ovary, uterus, cervix and vagina in females
Postmortem examinations (offspring):
Offspings were sacrificed to determined presence or absence of abnormality on the external surface
Statistics:
Fisher's direct probability test 1) was carried out on frequency of type of sex cycle, mating rate, conception rate, morphological abnormality frequency of babiesAccording to histopathological examination findings, the Mann-Whitney U test 2)) shows the grade-separated data, and the total value of the positive grade is obtained by Fisher's direct probability one-sided test 3) between the control group and each test substance administration group Significant difference test was carried out. For the other data, we tested the uniformity of variance of each group by Bartlett method 4) , with the value obtained for each individual, or the average value for each litter as one sample . If the variance is uniform, a one-way analysis of variance 4) was performed, and when significance was observed between the groups, multiple comparisons were performed according to the Dunnett method 5) . On the other hand, Kruskal-Wallis 6) rank test is performed when the variance is 0 in any group and when the variance is not uniform, and if significance is observed between the groups.
Reproductive indices:
Conception rate was determined
Offspring viability indices:
No data available
Clinical signs:
no effects observed
Description (incidence and severity):
Nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed Salivation was observed in each administration group of the test substance.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
In males, one patient in the 160 mg / kg dose group died on the 60th day of administration.
In females, there were no deaths or moribund
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference between the control group and each test group administered group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males no change in food consumption were observed, however in females no change in food consumption uring the period before mating, but significantly lower values than control group, after pregnancy and during the nursing stage
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes. There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormality was observed in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group. Unilateral seminal vesicle atrophy was observed in each group including the control group. no avnormality were seenon seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina.

Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hair loss was observed in all the treated animals
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant difference were observed in estrus cycle after administration in each group including the control group
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant difference between the conception rate, number of estrus periods in control and treated groups at 10, 40 and 160mg/kg. There was no significant difference in number of implantation between the control group and each test chemical treated groups.
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
not specified
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Deaths observed in 4 offspings of control group and 2 offsping of 10 mg / kg administration group, respectively. In addition, 3 out of 6 cases in the 40 mg / kg administration group and 22 cases out of 27 cases in the 160 mg / kg administration group were judged as stillbirth
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no significant difference between male and female between the control group and each test group administered group at any time.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
At necropsy of born pups, morphological changes including malformations and mutations were observed in born pups 1-2 in each group including the control group.
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
histopathology: non-neoplastic
Critical effects observed:
not specified
Key result
Reproductive effects observed:
not specified
Treatment related:
not specified
Conclusions:
From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.
Executive summary:

In the present study, one generation reproductive toxicity test of test chemical was carried out and the toxicity of reproductive to male and female animals of test chemical was examined. In the study, animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed. The results of the parenteral examination revealed that, no mortality in treated animals at 10, 40 and 160mg/kg/day for 10 weeks,including mating period, the clinical signs examination of animlas shows, nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed. Salivation was observed in each administration group of the test substance.No test chemical related chnages on food consumption and body weight changes were observed. Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes.  There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group. There was no change depending on the dose of the test substance in reproductive organsof male and females. The histopathological study relealed no abnormality in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group.  Unilateral seminal vesicle atrophy was observed in each group including the control group. No abnormality were seen on seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina. The fetal examination results revealed, deaths in 4 offspings of control group and 2 offsping of 10 mg / kg administration group, respectively. In addition, 3 out of 6 cases in the 40 mg / kg administration group and 22 cases out of 27 cases in the 160 mg / kg administration group were judged as stillbirth, but no clinical signs of toxicity on survived pups. There was no significant difference in body weight in male and female  between the control group and each test group administered group at any time. No gross pathological changes were observed, At necropsy of born pups, morphological changes including malformations and mutations were observed in  born pups 1-2 in each group including the control group. From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
160 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The data is K2 level and provides robbust summary
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The Reproductive Toxicity Data:

Study 2:

In the present study, one generation reproductive toxicity test of test chemical was carried out and the toxicity of reproductive to male and female animals of test chemical was examined. In the study, animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed. The results of the parenteral examination revealed that, no mortality in treated animals at 10, 40 and 160mg/kg/day for 10 weeks,including mating period, the clinical signs examination of animlas shows, nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed. Salivation was observed in each administration group of the test substance.No test chemical related chnages on food consumption and body weight changes were observed. Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes.  There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group. There was no change depending on the dose of the test substance in reproductive organsof male and females. The histopathological study relealed no abnormality in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group.  Unilateral seminal vesicle atrophy was observed in each group including the control group. No abnormality were seen on seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina. The fetal examination results revealed, deaths in 4 offspings of control group and 2 offsping of 10 mg / kg administration group, respectively. In addition, 3 out of 6 cases in the 40 mg / kg administration group and 22 cases out of 27 cases in the 160 mg / kg administration group were judged as stillbirth, but no clinical signs of toxicity on survived pups. There was no significant difference in body weight in male and female  between the control group and each test group administered group at any time. No gross pathological changes were observed, At necropsy of born pups, morphological changes including malformations and mutations were observed in  born pups 1-2 in each group including the control group. From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.

Study 3:

One-Generation Reproduction Toxicity Test of test chemical in Rats was performed to estimate the reproductive toxicity profile. In the present study dose levels of 0, 12.5 ,50 and 200mg/kg used for the 10 weeks of exposure (71 dose administration) including mating period of 3 weeks. The treated animals observed for general condition, body weight measurement and food intake. Red blood cell count and white blood cell count was also measured. Estrus cycle during exposure was estimated. The offsprings were examined for number of births, general condition, number of deceased offspings, litter count, body weight, sex ratio and survival rate were measured.The postmortem of parenteral animals was carried out to examine major thoracic abdominal organs including the pituitary gland, stomach, adrenal glands, testis, epididymis, coagulated glands, seminal vesicles, prostate,ovaries, uterus and vagina. All surviving infants were necropsied by sacrificing all cases by ether inhalation on the 21th birth. At that time, the organ in which abnormality was observed. The dead child was autopsy examined for the presence or absence of abnormality on the external surface. The results of the parenteral observations revealed, transient salivation after test chemical administration in the 200 mg / kg group in both males and female. One case in the control group died due to poor feeding due to irregular occlusion. In addition, each one of 12.5 and 50 mg / kg administration group was moribund slaughtered or died by myeloid leukemia, but neither was caused by administration of the test substance. No death or moribund was observed in other treated animals. In test chemical treated males, suppression of body weight gain was observed at the end of treatment  and in females, suppression of body weight gain was observed between the early stage of administration of 200 mg / kg administration group and the middle stage of pregnancy to postpartum nursing, but the effects of administration was not observed in food intake. No change in food consumption were seen at any dose levels at 12.5, 50 and 200mg/kg/day compared to control animals.  The increase in the number of red blood cells(RBC) in the administration group of 50 mg / kg or more, no change related to the administration of the test substance was observed. At necropsy, hyperplasia of the forestomach mucosa was observed in the 200 mg / kg dose group.. In the 200 mg / kg administration group, attenuation of the degree of periportal fatty liver in the liver was also observed. For the liver, swelling was also observed at autopsy, but no distinct tissue change associated with swelling was observed. Histopathological examination of tissues revealed, no change due to administration of the test substance. In addition, changes in the pituitary, testis, epididymis, coagulated glands, seminal vesicles and prostate did not occur in any of the administration groups. In females, hyperplasia of squamous epithelium of forestomachial mucosa, but adrenal gland, pituitary gland, ovary , Uterus, uterine cervix and vagina, no change due to administration of the test substance was observed. The observations of neonates revealed, no mortality at all dose levels at 12.5,50 and 200mg/kg/day. No clinical signs of toxicity were seen at all dose levels at 12.5, 50 and 200mg/kg/day. The body weight of the 200 mg / kg administration group decreased from birth date to 21 days after birth, and growth inhibition ranging from fetal stage to nursing stage was observed. The frequency of things with external malformations such as the tail, short tail, short bending tail or small eye slightly increased in the 200 mg / kg administration group. In the 200 mg / kg administration group, abnormalities in the testis and hypoplasia of the spleen and visceral malformation of the diaphragmatic hernia were also observed. From the observations and results, the NOAEL for parents and neonates was considered to be 200mg/kg/day and 50mg/kg/day respectively.

Study 4:

One generation reproductive toxicity study of test material was performed according to OECD Guideline 415. The test material mixed with diet in dose concentration 0, 100, 500 and 2500 ppm and adminstered to groups of 25 rats per sex per group. Males were treated for 10 weeks and during the mated period and females for at least two weeks before mating until sacrifice. Body weights were recorded weekly during gestation period and at days 1, 4, 7, 14 and 21 during lactation period. Food consumption was measured weekly during experimental period. Food intake was recorded on presumed gestation days 7, 14 and 20 and on lactation days 4, 7, 14, 18 and 21. At birth, the number of pups born, sex and body weight of individual pups on days 1 and 4 were recorded. After standardization of litter size to 8 pups, pups were weighed individually on days 7, 14 and 21 of lactation. On day of weaning sacrifice (day 21), only one randomly selected pup per sex per litter was submitted to macroscopic examination. Fertility index for dams, sires and pup survival index were calculated. On completion of the gross pathology examination, the following tissues and organs were preserved in 10% neutral buffered formalin and submitted to microscopic examination: grossly abnormal tissues, ovaries, uterus, vagina, testes, epididymides, seminal vesicles, prostate, coagulating glands, pituitary gland and target organs of all P animals. The number of corpora lutea and implantation sites were recorded for all the dams. Histopathological examination of the parents was initially restricted to preserved organs from control and high dose group animals.

There were significant lower body weights from control at high dose in males from weeks 2 to 6 and at week 8 associated with a significant lower food intake at weeks 2 and 8 at high dose in males. There was also a significant decrease in bodyweights at day 20 of the gestation period in females of the high dose group, also observed at days 4, 7 and 14 of lactation period. A statistically significant increase in relative kidney weights at mid dose in males and at high dose for males and females was observed. The increased kidney weights observed in males and females were minimal in nature (<12%) and were not associated with microscopic changes, hence considered as toxicologically insignificant changes. A statistically significant increase in relative epididymides was also recorded at high dose in males but with no related microscopic changes in left epididymides. Further, there were no corresponding change in the weight of right cauda epididymides and epididymal sperm count therefore the increased epididymal weights were considered as incidental changes.There were no test item related changes in sperm motility, cauda epididymal sperm counts and sperm morphology parameters. However, a statistically significant increase in percentage of abnormal sperms was observed in high dose males but this observed change was within the historical control data. Moreover, no fertility parameters were affected and there were no changes in the testes or epididymis grossly or histopathologically therefore, considered incidental and not related to the treatment. There were no test item related gross findings in males and females. Only single incidences of several gross findings observed in different groups were considered as incidental without any relation to test item administration. There were no test item related microscopic changes in males and females. All single or few incidences of microscopic findings observed in males and females were considered as incidental findings. The mean number and weight of male, female and total pups per litter at all the doses tested were unaffected by treatment. No treatment-related changes were observed in the data of pups up to lactation day 21 at all the doses tested. HenceNo Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be2500 ppm which is equivalent to 153.8 mg/kg Bwt/day for males and 393.6 mg/kg Bwt/day for females,.When male and femalerats were treated withtest chemical orallyover one generation.

Effects on developmental toxicity

Description of key information

The Developmental Toxicity Data :

In the present study, developmental toxicity test of test chemical was carried out and the developmental toxicity profile to male and female animals of test chemical was examined. In the study, animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed. The results of the parenteral examination revealed that, no mortality in treated animals at 10, 40 and 160mg/kg/day for 10 weeks,including mating period, the clinical signs examination of animlas shows, nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed. Salivation was observed in each administration group of the test substance.No test chemical related chnages on food consumption and body weight changes were observed. Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes.  There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group. There was no change depending on the dose of the test substance in reproductive organsof male and females. The histopathological study relealed no abnormality in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group.  Unilateral seminal vesicle atrophy was observed in each group including the control group. No abnormality were seen on seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina. The maternal developmental toxicity includes, Implantation number in treated animals was equivalent to control animals. The litter size was significantly low value in 160mg/kg/day group compared to control group. No change in pregnancy duration were seen in treated females. No abnormality in labor condition was observed, and there was no effect on birth rate. The examination of pups revealed, body weight at the postnatal day 21 was slightly lower in the 160 mg / kg administration group. Number of live pups examined reduced in highest dosed group. There was no effect of administration on sex ratio. The litter size after adjustment of litter size was decreased in 160mg/kg/day group. In 160mg/kg /day group the postnatal survival declines comapared to control.No morphological changes including external, skeletal and visceral malformations where seen in treated animals. From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Data is from Ministry of Health, Labor and Welfare report
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Principles of method if other than guideline:
Developmental toxicity test of test chemical was carried out in Sprague-Dawley rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley strain (Crj: CD) rats
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Tsukuba breeding center, Charles River Japan Co., Ltd.
- Age at study initiation: No data available
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: housed in a metallic wire mesh floor cage in a breeding room
- Use of restrainers for preventing ingestion (if dermal): yes/no: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): tap water, provided ad libitum
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1 ° C
- Humidity (%): 50 to 65%
- Air changes (per hr): about 15 times / hour
- Photoperiod (hrs dark / hrs light): lighting for 12 hours
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
CMC (carboxymethyl cellulose)
Remarks on MMAD:
No data available
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution was prepared by mixing the test chemical in vehicle with a magnetic stirrer

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: No data available
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): No data available
- Purity:No data available
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data available
Details on mating procedure:
Matings were conducted in males and females 1: 1 within the same group for a period of 3 weeks, and confirming the presence of sperm in the vaginal smear and confirming the vaginal plug every morning.
Duration of treatment / exposure:
10 weeks (71 days of administration)
Frequency of treatment:
Daily
Duration of test:
No data available
Remarks:
0 (vehicle control)= 25male and 25female
10mg/kg=25male and 25female
40mg/kg=25male and 25female
160 mg/kg=25male and 25female
No. of animals per sex per dose:
0 (vehicle control)= 25male and 25female
10mg/kg=25male and 25female
40mg/kg=25male and 25female
160 mg/kg=25male and 25female
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed.
Maternal examinations:
Parent animals were observed for mortality, general condition, change in weight, food intake, estrus cycle assessment. Animals were sacrificed to determined presence or absence of abnormalities of major organs thoracic abdomen, including the pituitary glands, stomach, testis, epididymis, coagulated glands, seminal vesicles, and prostate in males and pituitary, stomach, ovary, uterus, cervix and vagina in females
Ovaries and uterine content:
Estrus cycle were determined before 2 weeks of start of mating confirmation day, two weeks before the start of administration and two weeks after the start of the administration.
Fetal examinations:
Number of births, litter count and weight measurement. Offspings were sacrificed to determined presence or absence of abnormality on the external surface
Statistics:
Fisher's direct probability test 1) was carried out on frequency of type of sex cycle, mating rate, conception rate, morphological abnormality frequency of babiesAccording to histopathological examination findings, the Mann-Whitney U test 2)) shows the grade-separated data, and the total value of the positive grade is obtained by Fisher's direct probability one-sided test 3) between the control group and each test substance administration group Significant difference test was carried out. For the other data, we tested the uniformity of variance of each group by Bartlett method 4) , with the value obtained for each individual, or the average value for each litter as one sample . If the variance is uniform, a one-way analysis of variance 4) was performed, and when significance was observed between the groups, multiple comparisons were performed according to the Dunnett method 5) . On the other hand, Kruskal-Wallis 6) rank test is performed when the variance is 0 in any group and when the variance is not uniform, and if significance is observed between the groups.
Indices:
Conception rate was determined
Historical control data:
No data available
Clinical signs:
no effects observed
Description (incidence and severity):
Nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed Salivation was observed in each administration group of the test substance.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
In males, one patient in the 160 mg / kg dose group died on the 60th day of administration.
In females, there were no deaths or moribund
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference between the control group and each test group administered group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males no change in food consumption were observed, however in females no change in food consumption uring the period before mating, but significantly lower values than control group, after pregnancy and during the nursing stage
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes. There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was no change depending on the dose of the test substance in reproductive organsof male and females. Abnormality was not found in the reproductive organs of the 160 mg / kg administration group.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormality was observed in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group. Unilateral seminal vesicle atrophy was observed in each group including the control group. no avnormality were seenon seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina.

Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hair loss was observed in all the treated animals
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Implantation number in treated animals was equivalent to control animals
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
The litter size was significantly low value in 160mg/kg/day group compared to control group.
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No change in pregnancy duration were seen in treated females.
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No abnormality in labor condition was observed, and there was no effect on birth rate
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
behaviour (functional findings)
gross pathology
histopathology: non-neoplastic
maternal abnormalities
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
changes in pregnancy duration
other: labor condition was normal no effect on birth rate
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight at the postnatal day 21 was slightly lower in the 160 mg / kg administration group
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Number of live pups examined reduced in highest dosed group
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect of administration on sex ratio
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
The litter size after adjustment of litter size was decreased in 160mg/kg/day group
Changes in postnatal survival:
effects observed, treatment-related
Description (incidence and severity):
In 160mg/kg /day group the postnatal survival declines comapared to control
External malformations:
no effects observed
Description (incidence and severity):
No morphological changes including malformations where observed
Skeletal malformations:
no effects observed
Description (incidence and severity):
No morphological changes including malformations where observed
Visceral malformations:
no effects observed
Description (incidence and severity):
No morphological changes including malformations where observed
Other effects:
no effects observed
Description (incidence and severity):
No growth inhibition was observed in the administration group of 40 mg / kg or less
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
external malformations
skeletal malformations
visceral malformations
other: No growth inhibition
Key result
Developmental effects observed:
not specified
Conclusions:
From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.
Executive summary:

In the present study, developmental toxicity test of test chemical was carried out and the developmental toxicity profile to male and female animals of test chemical was examined. In the study, animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed. The results of the parenteral examination revealed that, no mortality in treated animals at 10, 40 and 160mg/kg/day for 10 weeks,including mating period, the clinical signs examination of animlas shows, nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed. Salivation was observed in each administration group of the test substance.No test chemical related chnages on food consumption and body weight changes were observed. Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes.  There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group. There was no change depending on the dose of the test substance in reproductive organsof male and females. The histopathological study relealed no abnormality in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group.  Unilateral seminal vesicle atrophy was observed in each group including the control group. No abnormality were seen on seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina. The maternal developmental toxicity includes, Implantation number in treated animals was equivalent to control animals. The litter size was significantly low value in 160mg/kg/day group compared to control group. No change in pregnancy duration were seen in treated females. No abnormality in labor condition was observed, and there was no effect on birth rate. The examination of pups revealed, body weight at the postnatal day 21 was slightly lower in the 160 mg / kg administration group. Number of live pups examined reduced in highest dosed group. There was no effect of administration on sex ratio. The litter size after adjustment of litter size was decreased in 160mg/kg/day group. In 160mg/kg /day group the postnatal survival declines comapared to control.No morphological changes including external, skeletal and visceral malformations where seen in treated animals. From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The data is K2 level and provides robbust summary
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

The Developmental Toxicity Data :

Study 2:

In the present study, developmental toxicity test of test chemical was carried out and the developmental toxicity profile to male and female animals of test chemical was examined. In the study, animals were orally administered from 10 weeks of age including mated up to 3 weeks. The males were necropsied after one week passed after the mating period. Females were allowed to spontaneously deliver after mating and necropsied with their babies on nursing 21th. Both males and females continued to be administered until the day before necropsy, during which the general condition of the parent animal, weight gain and changes in food intake were observed, and at the same time the reproductive ability, including delivery and lactation of the parent animal, and weaning of the infant was observed. The results of the parenteral examination revealed that, no mortality in treated animals at 10, 40 and 160mg/kg/day for 10 weeks,including mating period, the clinical signs examination of animlas shows, nasal discharge, eyelid ptosis or closed eyes, in group administered with 40 mg / kg or more but not in 160mg/kg group , lacrimation in 160 mg / kg administration group was transiently observed. Salivation was observed in each administration group of the test substance.No test chemical related chnages on food consumption and body weight changes were observed. Spontaneous locomotor reduction and salivation were observed transiently after administration and no abnormality was observed in both treated males and femlaes.  There was a decrease in locomotor activity, eyelid ptosis or closed eyes, and nasal discharge in group administered with 40 mg / kg or more, lacrimation in 160 mg / kg administration group. There was no change depending on the dose of the test substance in reproductive organsof male and females. The histopathological study relealed no abnormality in pituitary gland. In males, in each case in the 40 mg / kg and 160 mg / kg administration groups, the spermatogonia of the spermatogenic cells in the 14 th stage of the spermatogenesis cycle mild degeneration was observed, and cell debris was observed in the lumen of the epididymis in the example of 40 mg / kg administration group.  Unilateral seminal vesicle atrophy was observed in each group including the control group. No abnormality were seen on seminal vesicle and Coagulated glands In females, no abnormality was observed in the pituitary gland, stomach, ovary, uterus, cervix and vagina. The maternal developmental toxicity includes, Implantation number in treated animals was equivalent to control animals. The litter size was significantly low value in 160mg/kg/day group compared to control group. No change in pregnancy duration were seen in treated females. No abnormality in labor condition was observed, and there was no effect on birth rate. The examination of pups revealed, body weight at the postnatal day 21 was slightly lower in the 160 mg / kg administration group. Number of live pups examined reduced in highest dosed group. There was no effect of administration on sex ratio. The litter size after adjustment of litter size was decreased in 160mg/kg/day group. In 160mg/kg /day group the postnatal survival declines comapared to control.No morphological changes including external, skeletal and visceral malformations where seen in treated animals. From the observation and results, the NOAEL for P0 generation and F1 generation was considered to be 160mg/kg/day and 40mg/kg/day respectively.

Study 3:

In the present study, developmental toxicity Test of test chemical in Rats was performed to screen the developmental toxicity profile. In the study dose levels of 0, 12.5 ,50 and 200mg/kg used for the 10 weeks of exposure (71 dose administration) including mating period of 3 weeks. The treated animals observed for general condition, body weight measurement and food intake. Red blood cell count and white blood cell count was also measured. Estrus cycle during exposure was estimated. The offsprings were examined for number of births, general condition, number of deceased offspings, litter count, body weight, sex ratio and survival rate were measured.The postmortem of parenteral animals was carried out to examine major thoracic abdominal organs including the pituitary gland, stomach, adrenal glands, testis, epididymis, coagulated glands, seminal vesicles, prostate,ovaries, uterus and vagina. All surviving infants were necropsied by sacrificing all cases by ether inhalation on the 21th birth. At that time, the organ in which abnormality was observed. The dead child was autopsy examined for the presence or absence of abnormality on the external surface. The results of the parenteral observations revealed, transient salivation after test chemical administration in the 200 mg / kg group in both males and female. One case in the control group died due to poor feeding due to irregular occlusion. In addition, each one of 12.5 and 50 mg / kg administration group was moribund slaughtered or died by myeloid leukemia, but neither was caused by administration of the test substance. No death or moribund was observed in other treated animals. In test chemical treated males, suppression of body weight gain was observed at the end of treatment  and in females, suppression of body weight gain was observed between the early stage of administration of 200 mg / kg administration group and the middle stage of pregnancy to postpartum nursing, but the effects of administration was not observed in food intake. No change in food consumption were seen at any dose levels at 12.5, 50 and 200mg/kg/day compared to control animals.  The increase in the number of red blood cells(RBC) in the administration group of 50 mg / kg or more, no change related to the administration of the test substance was observed. At necropsy, hyperplasia of the forestomach mucosa was observed in the 200 mg / kg dose group.. In the 200 mg / kg administration group, attenuation of the degree of periportal fatty liver in the liver was also observed. For the liver, swelling was also observed at autopsy, but no distinct tissue change associated with swelling was observed. Histopathological examination of tissues revealed, no change due to administration of the test substance. In addition, changes in the pituitary, testis, epididymis, coagulated glands, seminal vesicles and prostate did not occur in any of the administration groups. In females, hyperplasia of squamous epithelium of forestomachial mucosa, but adrenal gland, pituitary gland, ovary , Uterus, uterine cervix and vagina, no change due to administration of the test substance was observed. The examination of neonates revealed, no change on the survivability and no. of pup. However, the body weight of the 200 mg / kg administration group decreased from birth date to 21 days after birth but not in 50mg/kg and lower doses. External malformations such as the tail, short tail, short bending tail or small eye slightly increased in the 200 mg / kg administration group. Abnormalities in the testis and hypoplasia of the spleen and visceral malformation of the diaphragmatic hernia were also observed in 200mg/kg group. At the 200 mg / kg administration group growth inhibition ranging from fetal stage to nursing stage was observed. From the observations and results, the NOAEL for parents and neonates was considered to be 200mg/kg/day and 50mg/kg/day respectively.

Study 4:

The developmental toxicity study of test material was performed according to OECD guideline 414 on rabbits.Young adult nulliparous New-Zealand white strain female and male rabbits (Oryctolagus cuniculus) were used in study. The test material dissolved in 0.5% Carboxymethyl cellulose in dose concentration 0, 3, 10 and 50 mg/kg/day and adminstered by daily gavage through gestation day 6 to 28 to mated females (25/dose group).The preliminary range-finding study (0, 10, 60 and 300 mg/kg/day) was performed, Based on preliminary range-finding study findings, 0, 3, 10 and 50 mg/kg/day were selected for the main study. Animals were observed twice daily for moribundity and mortality. Individual clinical signs were recorded at least once a day during the treatment period and once daily during the pre- and post-treatment periods. Individual body weights were recorded on gestation days 0, 3, 5, 8, 11, 14, 17, 20, 23, 26, 29 and 30. Food consumption per cage of animals were measured over the following periods during gestation: days 0-3, 3- 5, 5-8, 8-11, 11-14, 14-17, 17-20, 20-26, 26-29 and 29-30. On Day 29 of gestation, all the rabbits were sacrificed using intravenous injection of sodium thiopentone. Any gross pathological changes in all the visceral organs of dams were recorded. Organs with macroscopic findings and kidneys were preserved for possible histological evaluation. The ovaries, uteri and uterine contents were removed and examined to determine: the number of corpora lutea, the number of implantations, early and late resorptions, the weight of intact gravid uterus, the number and distribution of live foetuses, the number and distribution of intra-uterine dead foetuses, the individual foetal weight and sex, foetal abnormalities.

There were no maternal death or necropsy findings at any dose levels. There was a significant reduction in the body weight gain during the treatment period in the high dose group (50 mg/kg). The food consumption was comparable to the vehicle control group. The reduction in body weight during the treatment period was considered treatment related. One rabbit aborted in the high dose group, there were 2 non pregnant rabbits in control, 4 in low dose group, 3 in mid dose group and 4 in the high dose group. There was one complete resorption in mid dose group. At the end, at least 20 litters were observed in each of the dose groups. The maternal data parameters comprising of implantations, early and late resorptions, pre and post-implantation loss in all the treatment groups were comparable to the vehicle control group. The mean number of corpora lutea, implantation and live foetus were significantly lower in high dose group (50 mg/kg body weight/day) when compared with the control group. Observed decrease in corpora lutea at 50 mg/kg body weight/day is considered as biological variation because the treatment was initiated after the implantation (gestation day 6). Therefore, the decrease observed in the absolute uterine weight, implantation and live foetus reported at this dose level are also considering as biological variation as these observations are directly correlated with the decrease in the number of the corpora lutea. Hence No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 50 mg/kg/day,when female rabbits were treated withtest materialorally.

 

Justification for classification or non-classification

Based on the data available for test chemical, the test chemical does not exhibit reproductive and developmental toxicity by oral route of administration. Hence the test chemical is not likely to classify as a reproductive/ developmentat toxicant as per the criteria mentioned in CLP regulation.