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Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Study:

In three generation reproductive toxicity study, Total 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group.90 animals of each sex were used in control group. The test chemical was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation. In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2ageneration. Same procedure was used to produce F2b generation. After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues. The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and lower dose group. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group. Also No dose related effects were observed on gross deformities in each generation. The only significant histopathological changes noted in F3ageneration were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female).Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen (p<0.001 for male and p<0.00001 for female). Hence the NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested. While LOAEL for offspring in F3a generation was considered at 5mg/kg bw/day when male and female Fischer 344 rats were treated with the test chemical orally.

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 based on information from various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
equivalent or similar to
Guideline:
other: OECD 414 (Pre-Natal Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The above experiments were performed to analyse and evaluate any toxicity caused to the reproduction and developmental parameters in the test animals by the test chemical.
GLP compliance:
not specified
Justification for study design:
No Data Available
Specific details on test material used for the study:
Details on test material
- Name of test material (as cited in study report): 4,4'-Bis(dimethylamino)-4''-(methylamino)trityl alcohol
- Molecular formula (if other than submission substance): C24-H29-N3-O
- Molecular weight (if other than submission substance): 375.513 g/mol
- Substance type: Organic
- Physical state: No Data Available
- Impurities (identity and concentrations): No Data Available
Species:
other: Study 2, 3, 4, 5: Rat
Strain:
other: Study 2: Fischer 344 Study 3 and 4: Crj: CD (SD) Study 5: Sprague-Dawley
Details on species / strain selection:
No Data Available
Sex:
male/female
Details on test animals and environmental conditions:
Study 2: No Data Available
Study 3: Age at study initiation of dosing: 10 weeks old
Study 4: No Data Available
Study 5: After 2 weeks of acclimatization, Sprague–Dawley healthy virgin female rats(11–12 weeks old) from Guangdong Medical Laboratory Animal Center were mated with males (13–14 weeks old) of the same rat strain from the same source in the late afternoon. Vaginal smears were taken the following morning until a positive identification of mating was made. The appearance of sperm in a vaginal smear or the presence of a vaginal plug was designated day 0 of gestation (GD 0). Sperm positive females were assigned to each study group based on body weight. The distribution of males to females was uniform among groups.The animals were individually housed in suspended stainless steel group cages,each with a wire mesh floor and front. The temperature in the animal room was maintained at 21–26 C, and relative humidity was 40–70%, with 12 air changes per hour and a 12 h light–dark cycle. Food and tap water were available ad libitum.
Route of administration:
other: Study 2: oral:feed, Study 3, 4, 5: oral:gavage
Vehicle:
other: Study 2: Feed; Study 3: 0.5 % Methylcellulose aqueous solution; Study 4: 0.5 w/v% CMC-Na solution containing 0.1 w/v% Tween 80; Study 5: unchanged (no vehicle)
Details on exposure:
Study 2: No data Available

Study 3: PREPARATION OF DOSING SOLUTIONS: Dose were prepared by suspending test chemical in 0.5 % Methylcellulose aqueous solution at 0, 1.6, 8 and 40 mg/kg bw.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 % Methylcellulose aqueous solution
- Concentration in vehicle: 0, 1.6, 8 and 40 mg/kg bw.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required):
- Purity: No Data Available

Study 4: PREPARATION OF DOSING SOLUTIONS: Test chemical suspended in 0.5 w/v% CMC-Na solution containing 0.1 w/v% Tween 80 at 0, 40, 200 and 1000 mg/kg bw

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 w/v% CMC-Na solution containing 0.1 w/v% Tween 80
- Concentration in vehicle: 0, 40, 200 and 1000 mg/kg bw
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity: No Data Available

Study 5: The test chemical suspensions at 1, 8, and 16 mg/mL were prepared in 1% Tween-80 (v/v, distilled water) within 6 h prior to each dosing.
Details on mating procedure:
Study 2:
- M/F ratio per cage: No data available
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy:No data available
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.No data available
- Further matings after two unsuccessful attempts: [no / yes (explain)]No data available
- After successful mating each pregnant female was caged (how): single

- Any other deviations from standard protocol:No data available

Study 3 and 4: No data available

Study 5:
After 2 weeks of acclimatization, Sprague–Dawley healthy virgin female rats(11–12 weeks old) from Guangdong Medical Laboratory Animal Center were mated with males (13–14 weeks old) of the same rat strain from the same source in the late afternoon. Vaginal smears were taken the following morning until a positive identification of mating was made. The appearance of sperm in a vaginal smear or the presence of a vaginal plug was designated day 0 of gestation (GD 0). Sperm positive females were assigned to each study group based on body weight. The distribution of males to females was uniform among groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Study 2, 3, 4: No Data Available
Study 5: For homogeneity analysis, samples (from the top, middle, and bottom portions of the formulations) were collected from the test chemical dosing formulations at the concentrations of 1 and 16 mg/mL prepared for use on the first dosing day. The mid layer homogeneity results also served as concentration verification results. Acceptance criteria for potency of the initial formulation preparation should be within ±10%= of the nominal concentration. All the homogeneity results and concentration verification results met the acceptance criteria.
Duration of treatment / exposure:
Study 2: Total: 280-294
F0 generation :at least 80days
F1b generation:100-140 days
F2b generation:100-140 days
F3a generation:up to weaning

Study 3 and 4: Male: 42 day
Female: 41 - 48 days (from 14 days before mating to day 4 of lactation)

Study 5: The test chemical was administered to the test animals to from day 6–18 of gestation.
Frequency of treatment:
Study 2 to 5: Daily
Details on study schedule:
Study 2:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
After at lest 80 daysfrom start of study, In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation
- Selection of parents from F1 generation when pups were [...] days of age.:
At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2a generation. Same procedure was used to produce F2b generation
- Age at mating of the mated animals in the study: [...] weeks
After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation.

Study 3, 4 and 5: No Data Available
Remarks:
Study 2: 0, 5,15 and 30 mg /kg bw /day
Study 3: 0, 1.6, 8 and 40 mg /kg bw /day
Study 4: 0, 40, 200 and 1000 mg /kg bw /day
Study 5: 0 (control), 10, 40, 120, and200 mg/kg/day.
No. of animals per sex per dose:
Study 2:
Total : 450 animals
Control group:180 animals
Male :90 animals
female 90 animals
treated group:270 animals
5mg/kg bw/day:45 male and 45 female
15mg/kg bw/day:45 male and 45 female
30mg/kg bw/day:45 male and 45 female

Study 3:
Total: 106 animals
0 mg/kg/day: 7 male, 12 female
1.6 mg/kg/day: 12 male, 12 female
8 mg/kg/day: 12 male, 12 female
40 mg/kg/day: 7 male, 12 female
Recory group:
0 mg/kg/day: 5 male, 5 female
40 mg/kg/day: 5 male, 5 female

Study 4:
Total: 106
0 mg/kg/day: 7 male, 12 female
40 mg/kg/day: 12 male, 12 female
200 mg/kg/day: 12 male, 12 female
1000 mg/kg/day: 7 male, 12 female
Recory group:
0 mg/kg/day: 5 male, 5 female
1000 mg/kg/day: 5 male, 5 female

Study 5: eight sperm positive females/group
Control animals:
yes, concurrent vehicle
Details on study design:
Study 2, 3 and 4: No data
Study 5: To determine the dose range, eight sperm positive females/group were given the test chemical by gavage daily from GD 6–15 at dosages of 0 (control), 10, 40, 120, and 200 mg/kg/day. The results show that the administration of 200 mg/kg/day caused maternal death (2 of 8 treated pregnant rats). The post-implantation loss was found at 120–200 mg/kg/day. Minor changes of body weight were revealed at 40 mg/kg/day. No evidence of adverse effects was found at 10 mg/kg/day.
Based on these results, 30 sperm positive females/group were administered the test chemical by gavage at 0 (control), 10, 80, and 160 mg/kg/day. Dosing occurred once daily in the morning from GD6–15. The dosing volume was 10 mL/kg. Initial doses were based on GD6 weight and adjusted every 3 days throughout the treatment period. A control group received the vehicle under the same conditions. Caesarean sections were performed on GD20, and fetuses were examined for developmental effects.
Positive control:
Study 2 to 5: No data available
Parental animals: Observations and examinations:
Study 2: CAGE SIDE OBSERVATIONS: No data
- Time schedule: No Data
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No Data

BODY WEIGHT: Yes
- Time schedule for examinations: No Data

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OTHER:No data

Study 3 and 4: Survival, clinical sign, body weight, food consumption and urineanalysis, FOB, Urinalysis (Male only), were examined.

Study 5: Animals were observed twice daily before and after treatment for signs of overt toxicity, changes in general appearance, and mortality. Sperm positive females were weighed on GD0, 3, 6, 9, 12, 15, and 20. Final GD20 body weights were corrected for gravid uterine weights. Food consumption was measured on GD0, 6, 9, 12, 15, and 19. Water consumption was monitored daily. Three females in each group were euthanized by deep anesthesia with sodium pentobarbital followed by exsanguination on GD7 and GD16, respectively. At least 2 mL blood was collected from each animal via the abdominal aorta under deep anesthesia. At least 1 mL amniotic fluid were collected from each dam euthanized on GD16. Blood samples were placed in iced water until they were centrifuged to prepare the plasma samples. All plasma and amniotic fluid samples were stored at 80 C until analysis. On GD20, the surviving mated females were euthanized by CO2 asphyxiation followed by exsanguination. The blood and amniotic fluid samples were collected from the first three pregnant animals necropsied in each group, as described above. All the study animals were examined for gross abnormalities. The livers of pregnant rats in the highest dose group and in the control group were fixed in 10% buffered formalin on GD20. A full histopathological examination was performed on hematoxylin- and eosin-stained tissue sections of the liver.
Oestrous cyclicity (parental animals):
Study 2 and 5: No data Available
Study 3and 4: Estrous cyclicity, copulation and implantation were examined.
Sperm parameters (parental animals):
Study 2 to 5: No Data Available
Litter observations:
Study 2:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]: No data available
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); for F1b and F2a generation one male and one female from each litter were seleted and 2 male and 2 female per litter for F3a generation were selected excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:yes
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, were observed

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]No data available

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:No data available

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:No data available
Study 3 and 4: Number of pups, number of live pups, sex ratio on days 0 and 4, clinical signs and body weight were examined.

Study 5: After necropsy on GD20, the uteri were removed and weighed. The number of total implantations and corpora lutea, the number and location of viable and
non-viable fetuses, early and late resorptions, and all externally visible fetal abnormalities were recorded. All live fetuses and placentas were weighed. The crown- rump length of all live fetuses was measured and their sex was determined. Uteri from females that appeared non-gravid and individual uterine horns without visible implantations were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. All fetuses were examined for external abnormalities. Approximately one-half of the fetuses in each litter were eviscerated (the livers and kidneys of these fetuses were weighed) and stained with Alcian blue and Alizarin red S for subsequent skeletal examination.he remaining fetuses were fixed in Bouin’s solution for soft tissue examination. The descriptions of the variations and malformations were in accordance with the terms for developmental abnormalities in common laboratory animals.
Postmortem examinations (parental animals):
Study 2: SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]No data available
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.] No data available

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]No data available

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.: yes in F3a generations were observed.

Study 3 and 4: Hematology, clinical chemistry, Organ weight, gross pathology and histopathology were examined.
Study 5: On GD20, the surviving mated females were euthanized by CO2 asphyxiation followed by exsanguination. The blood and amniotic fluid samples were collected from the first three pregnant animals necropsied in each group, as described above. All the study animals were examined for gross abnormalities. The livers of pregnant rats in the highest dose group and in the control group were fixed in 10% buffered formalin on GD20. A full histopathological examination was performed on hematoxylin- and eosin-stained tissue sections of the liver. After necropsy on GD20, the uteri were removed and weighed. The number of total implantations and corpora lutea, the number and location of viable and non-viable fetuses, early and late resorptions, and all externally visible fetal abnormalities were recorded. All live fetuses and placentas were weighed.
Postmortem examinations (offspring):
Study 2: SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: No data available

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]No data available

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively. yes in F3a generation.

Study 3 and 4: Gross pathology were examined.

Study 5: Uteri from females that appeared non-gravid and individual uterine horns without visible implantations were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. All fetuses were examined for external abnormalities. Approximately one-half of the fetuses in each litter were eviscerated (the livers and kidneys of these fetuses were weighed) and stained with Alcian blue and Alizarin red S for subsequent skeletal examination.he remaining fetuses were fixed in Bouin’s solution for soft tissue examination. The descriptions of the variations and malformations were in accordance with the terms for developmental abnormalities in common laboratory animals.
Statistics:
Study 2 to 5: No data Available
Reproductive indices:
Study 2: No data Available
Study 3 and 4: Fertility rat, gestation period, implantation index, live birth index, delivery index were examined.
Study 5: Implantation Index, Resorption Index, Uterine Index.
Offspring viability indices:
Study 2: No data Available
Study 3 and 4: Viability index on 0 and 4 day were examined.
Studt 5: Pup Viablility Index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female survival rats were observed. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.

Study 4: No clinical signs were observed in treated rats as compared to control.

Study 5: Perineal yellowish to brownish staining was noted in two high-dose females on GD11 and GD12, respectively. There were reddish substances on the tray under the two animals’ cages. Additionally, Alopecia was observed in seven females at 160 mg/kg/day, and it was first noted on GD9 or later. No other treatment-related clinical findings were noted.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Study 2: No dose related effect on mortality was observed
Study 3: When treated with 40 mg/kg bw, 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity.
No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control.
Study 4: No effect on survival of treated rats were observed at 40, 200 and 1000 mg/kg bw as compared to control.
Study 5: All females survived up to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 2: The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group.
Study 3: When treated with 40 mg/kg/day, decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats as compared to control.
Study 4: No change in body weight were observed in treated rats as compared to control.
Study 5: Average maternal body weight in each group before dosing (body weight of GD0, 3, 6) were similar, whereas body weights after dosing (GD9, 12, 15, and 20) in the 80 and 160 mg/kg/day dose group were significantly less than those in the control group.The body weight gain decreased in a dose-related manner during treatment period (GD6–15). Thus, the 80 mg/kg/day dose animals gained less than half the weight gain of the control group, while the 160 mg/kg/day dose rats lost weight. Weight gain was decreased, especially during the early period of treatment (GD 6–9).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, decrease in food consumption was observed in male and female rats as compared to control.
Study 4: No change in food consumption were observed in treated rats as compared to control.
Study 5: During the period of administration (GD6–15), significant decrease in maternal food consumption was observed at 80 and 160 mg/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, Increase in the PLT was observed in male and female rats as compared to control.
Study 4: No changes in hematological parameters were observed in treated male and female rats as compared to control.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat.
Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and Increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals.
Study 4: No changes in clinical chemisrty were observed in treated male and female rats as compared to control.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Study 3: No effect was observed in male rats as compared to control.
Study 4:No effect was observed in male rats as compared to control.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, Bradypnea, Prone position, Decrease in spontaneous activity, Incomplete eyelid opening and abnormal gait in died animals and no effect on surviving animals were observed.
Study 4: No changes in behavior test were observed in treated male and female rats as compared to control.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Study 3: No effect on organ weight of treated was male and female rats were observed as compared to control.
Study 4: No effect on organ weight of treated male and female rats were observed as compared to control.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extrimlly killed animals.

When treated with 40 mg/kg/day, Light violet aqueous content in alimentary tract in surviving male and female rats.

When treated with 8 mg/kg/day, Light violet aqueous content in stomach and cecum were observed in male rats (3/12) as compared to control.

Study 4: No gross pathological changes were observed in treated male and female rats as compared to control.

Study 5: No gross abnormalities were found in the dams at necropsy.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Study 3: When treated with 40 mg/kg/day, Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals. Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7) and Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals.

Study 4: Focal necrosis of mucosa and hyperplasia of gastric pits in the glandular stomach were observed in female rat at 200 and 1000 mg/kg bw.
The focal necrosis of mucosa is occasionally seen in the mucosa of the glandular stomach when the chemical compounds are administrated. Many of these induced changes have reversibility. focal necrosis of mucosa were not noted at the end of the recovery period, indicating reversibility. The hyperplasia of mucosal epithelium in the glandular stomach was considered to be a regeneration or restoration image against early epithelium disorder. The image of hyperplasia disappeared by complete repairing at the end of the recovery period.

Study 5: Upon histopathological examination of the maternal liver lesions in the highest-dose group and in the controls, there were no abnormal observations.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Study 3: No effect on Estrous cyclicity, copulation and inplantation were observed.
Study 4: No effect on Estrous cyclicity, copulation and inplantation were observed.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Study 3: No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .
Study 4: No effect on Reproductive performance of treated male and female rats were observed as compared to control.
Study 5: The number of corpora lutea per dam, number of implants per dam, and pre-implantation loss were not affected by LMG. The post-implantation loss was 59% at 160 mg/kg/day, whereas it was 8%, 7%, and 10% at 0, 10, and 80 mg/kg. Only the increase at the highest dose was statistically significant. At 160 mg/kg/day, most of the post-implantation loss (98%) consisted in resorptions. The incidence of dams with more than one resorption increased with the dose. In the 160 mg/kg/day dose group, 10 of 21 dams had completely resorbed litters, two of which were not visible macroscopically.
Study 2: Clinical signs:No data available
Mortality: No dose related mortality was observed,

Body weight and food consumption: The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group.

Test substance intake:No data available

Reproductive function: estrous cycle:No effect observed

Reproductive function: sperm measures:No data available

Reproductive performance:No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .

Organ weights:No data available

Gross pathology:No data available

Histopathology:No data available
other findings:No data available

Study 3: No Data Available
Study 4:No Data Available
Study 5: No Data Available
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effect observed on reproductive performance
Critical effects observed:
not specified
System:
other: Not Specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Description (incidence):
Study 2: No dose related mortality was observed,
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 2: The dose related effect in body weight was observed at dose 30mg/kg bw /day group compared to control and lower dose group
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
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Study 2: No effect observed on fertility index and stillborn animals ,sex ratio, number of pups per litter .
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effects on Reproductive Parameters.
Clinical signs:
no effects observed
Description (incidence and severity):
Study 3: No Clinical signs were observed in treated pups as compared to control.
Study 4: No Clinical signs were observed in treated pups as compared to control.
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
Study 3: No effect on suvival of pups were observed as compared to control.
Study 4: No effect on suvival of pups were observed as compared to control.
Study 5: Increased Mortality was observed at the highest dose group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Study 3: No effect on body weight of pups were observed as compared to control.
Study 4: No effect on body weight of pups were observed as compared to control.
Study 5: The test chemical induced a dose-related reduction in fetal body weight and placental weight which reached the statistical significance at 160 mg/kg/day
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
Description (incidence and severity):
Study 2: No dose related effects on incidence of gross deformities were noted
Study 3: No gross pathological changes were observed in treated pups as compared to control.
Study 4: No gross pathological changes were observed in treated pups as compared to control.
Study 5: The incidences of fetuses and litters with external abnormality in the treated groups were not significantly different from those in the control group. A few cases (1 or 2 per group) of anasarca, exencephaly, and hindlimb hyperextension were only observed at 80 and 160 mg/kg/day. Significantly increased incidences of fetuses and litters with visceral abnormality were detected only in the 160 mg/kg/day group compared with the control values. No specific visceral alteration was significantly different among the four groups. Some visceral abnormalities, such as misshapen cerebrum, enlarged ventricular chamber, small kidney, dilated ureter, small lung, and supernumerary lung lobe, were only observed in the treated groups. The test chemical induced a dose-related increased incidence of fetuses (litters) with skeletal abnormalities. The number of affected fetuses (litters) was statistically significant in the two higher-dose groups compared with the control. The predominant skeletal anomalies observed were bipartite ossification of the thoracic centrum. The fetal(litter)incidences of bipartite ossification of the thoracic centrum were significantly higher than control at 80 and 160 mg/kg/day, and were 33% (74%) and 69% (100%), respectively. At 160 mg/kg/day, the fetal and litter incidences of bipartite ossification of the lumbar centrum were 8% and 36%, also significantly higher than that of the control, in which no fetus with this abnormality was observed. Other skeletal abnormalities, such as detached rib cartilage, supernumerary thoracic centrum, absent lumbar centrum, supernumeary lumbar centrum, and misshapen sacral arch, were only observed in the dose groups, but were not significantly increased.
Histopathological findings:
not specified
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
Study 2: No dose related effects on incidence of gross deformities were noted
Study 5: Doses of 80 and 160 mg/kg/day induced fetal skeletal abnormalities. The major skeletal defects were bipartite ossification of the thoracic centrum. The test chemical at 160 mg/kg/day also induced marked embryolethality and visceral abnormalities.a dose level of 10mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for fetal developmental toxicity in rats.
Dose descriptor:
NOAEL
Generation:
other: F2b
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Remarks on result:
other: No effect observed at dose 30mg/kg bw
Critical effects observed:
not specified
System:
other: Not Specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
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:
not specified
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Study 2: Histopatological finding: Dose related trend for focal dilatation of the renal cortex and tubules ,for necrosis of the thymus and inverse dose-response relationship for red pulp haematopoietic cell proliferation of the spleen
Other effects:
not specified
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
No Data Available
Dose descriptor:
LOAEL
Generation:
other: F3a
Effect level:
5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Dose related effect observed
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/kg bw/day (nominal)
Organ:
spleen
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Reproductive effects observed:
no
Lowest effective dose / conc.:
30 mg/kg bw/day (nominal)
Treatment related:
no

Study 4:

Absolute and relative organ weights in rats treated orally with the test chemical in the combined repeated dose and reproductive/developmental toxicity screening test

Sex                      

Dose level (mg/kg)

Administration period

Recovery period

0

40

200

1000

0

1000

Male

Number of animals

5

5

5

5

5

5

Final body weight(g)

493.1 ± 35.9(7)a)

480.7 ± 33.6(12)

486.2 ± 27.2(12)

466.6 ± 28.1(7)

503.8 ± 12.5

510.0 ± 19.2

Absolute organ weight

 

 

 

 

 

 

Brain(g)

2.154 ± 0.079

2.122 ± 0.136

2.132 ± 0.061

2.064 ± 0.093

2.112 ± 0.030

2.168 ± 0.077

Thymus(mg)

359.2 ± 76.7

359.8 ± 105.9

334.0 ± 53.8

379.0 ± 39.2

368.2 ± 81.3

279.8 ± 52.8

Heart(g)

1.466 ± 0.171

1.528 ± 0.147

1.572 ± 0.156

1.648 ± 0.210

1.636 ± 0.145

1.636 ± 0.193

Liver(g)

12.088 ± 1.724

11.506 ± 0.575

12.304 ± 0.993

11.750 ± 0.792

13.082 ± 0.587

12.634 ± 0.882

Spleen(g)

0.826 ± 0.112

0.802 ± 0.087

0.756 ± 0.078

0.798 ± 0.099

0.782 ± 0.070

0.794 ± 0.145

Kidneys(g)

3.268 ± 0.364

3.172 ± 0.288

3.206 ± 0.320

3.002 ± 0.119

3.218 ± 0.133

3.376 ± 0.285

Adrenals(mg)

72.00 ± 15.34

65.94 ± 7.89

66.40 ± 7.42

66.04 ± 8.25

66.96 ± 7.48

64.50 ± 9.45

Testes(g)

3.339 ± 0.294(7)

3.440 ± 0.291(12)

3.472 ± 0.277(12)

3.554 ± 0.236(7)

3.284 ± 0.252

3.232 ± 0.631

Epididymides(g)

1.350 ± 0.085(7)

1.388 ± 0.083(12)

1.376 ± 0.082(12)

1.406 ± 0.089(7)

1.438 ± 0.121

1.340 ± 0.241

Relative organ weight

Brain(g%)

0.442 ± 0.035

0.452 ± 0.018

0.450 ± 0.032

0.444 ± 0.036

0.418 ± 0.013

0.426 ± 0.009

Thymus(mg%)

72.96 ± 11.71

76.58 ± 21.76

70.04 ± 7.72

81.10 ± 5.48

73.20 ± 16.87

54.90 ± 10.64

Heart(g%)

0.298 ± 0.026

0.328 ± 0.038

0.330 ± 0.016

0.354 ± 0.037

0.324 ± 0.034

0.320 ± 0.027

Liver(g%)

2.460 ± 0.166

2.458 ± 0.149

2.590 ± 0.155

2.520 ± 0.124

2.598 ± 0.134

2.476 ± 0.118

Spleen(g%)

0.170 ± 0.012

0.170 ± 0.019

0.160 ± 0.020

0.172 ± 0.030

0.156 ± 0.011

0.156 ± 0.025

Kidneys(g%)

0.666 ± 0.026

0.676 ± 0.048

0.678 ± 0.080

0.646 ± 0.060

0.638 ± 0.043

0.660 ± 0.042

Adrenals(mg%)

14.62 ± 2.23

14.12 ± 1.84

14.08 ± 2.48

14.18 ± 2.11

13.26 ± 1.28

12.60 ± 1.36

Testes(g%)

0.676 ± 0.030(7)

0.718 ± 0.079(12)

0.717 ± 0.075(12)

0.764 ± 0.077(7)

0.654 ± 0.039

0.634 ± 0.118

Epididymides(g%)

0.276 ± 0.022(7)

0.291 ± 0.025(12)

0.284 ± 0.019(12)

0.301 ± 0.032(7)

0.284 ± 0.024

0.262 ± 0.041

Female

Number of animals

5

5

5

5

5

5

Final body weight(g)

305.2 ± 26.7

314.4 ± 13.2

318.0 ± 22.2

314.2 ± 20.1

294.2 ± 31.1

299.0 ± 17.1

Absolute organ weight

Brain(g)

1.958 ± 0.044

1.940 ± 0.059

1.972 ± 0.052

2.008 ± 0.052

1.952 ± 0.048

1.990 ± 0.066

Thymus(mg)

223.2 ± 71.3

323.4 ± 54.4

303.6 ± 74.3

356.6 ± 46.3*

377.0 ± 111.5

335.2 ± 99.4

Heart(g)

1.028 ± 0.082

1.030 ± 0.037

1.078 ± 0.073

1.094 ± 0.070

0.912 ± 0.053

0.962 ± 0.040

Liver(g)

11.058 ± 0.861

10.332 ± 0.882

10.618 ± 0.689

10.824 ± 0.585

7.354 ± 0.986

7.922 ± 0.804

Spleen(g)

0.768 ± 0.094

0.624 ± 0.041

0.794 ± 0.063

0.902 ± 0.141

0.594 ± 0.065

0.522 ± 0.078

Kidneys(g)

2.212 ± 0.196

2.118 ± 0.151

2.386 ± 0.240

2.186 ± 0.222

1.986 ± 0.123

1.938 ± 0.075

Adrenals(mg)

81.30 ± 9.55

82.64 ± 4.70

79.46 ± 12.12

75.76 ± 16.53

77.86 ± 19.32

73.22 ± 10.90

Relative organ weight

Brain(g%)

0.646 ± 0.059

0.618 ± 0.026

0.618 ± 0.036

0.642 ± 0.039

0.668 ± 0.070

0.670 ± 0.056

Thymus(mg%)

72.96 ± 22.82

102.52 ± 13.98*

94.82 ± 18.35

113.64 ± 14.99**

127.10 ± 26.70

113.00 ± 35.65

Heart(g%)

0.336 ± 0.034

0.328 ± 0.011

0.338 ± 0.008

0.346 ± 0.009

0.314 ± 0.026

0.322 ± 0.008

Liver(g%)

3.632 ± 0.195

3.286 ± 0.249*

3.344 ± 0.180

3.444 ± 0.075

2.494 ± 0.086

2.646 ± 0.166

Spleen(g%)

0.254 ± 0.038

0.200 ± 0.014*

0.250 ± 0.020

0.286 ± 0.043

0.202 ± 0.008

0.176 ± 0.025

Kidneys(g%)

0.728 ± 0.033

0.672 ± 0.036

0.750 ± 0.043

0.694 ± 0.040

0.680 ± 0.060

0.650 ± 0.039

Adrenals(mg%)

26.70 ± 2.88

26.30 ± 1.17

25.08 ± 4.44

23.94 ± 3.94

26.52 ± 6.48

24.50 ± 3.87

a) Number of animals examied.

Values are expressed as Mean ± S.D.

Significantly different from 0 mg/kg group; * p <0.05, ** p <0.01

Histopathological findings in rats treated orally with the test chemical in the combined repeated dose and reproductive/developmental toxicity screening test

Sex

Organ Finding

Dose level (mg/kg)

Number of animals

Administration period

Recovery period

0

40

200

1000

0

1000

7

12

12

7

5

5

Male                          (Grade)

 Urinary bladder

<5>

<0>

<0>

<5>

<0>

<0>

Testis

<5>

<0>

<0>

<5>

<0>

<1>

Degeneration, seminiferous tubular epithelium, focal

1+

1

 

 

0

 

0

Degeneration, seminiferous tubular epithelium, diffuse

1+

0

 

 

0

 

1

Epididymis

<5>

<0>

<0>

<5>

<0>

<1>

Decrease, sperm

 

0

 

 

0

 

1

Seminal vesicle

<5>

<0>

<0>

<5>

<0>

<0>

Prostate

<5>

<0>

<0>

<5>

<0>

<0>

Cell infiltration, lymphocyte, interstitium

1+

1

 

 

2

 

 

Coagulating gland

<5>

<0>

<0>

<5>

<0>

<0>

Pituitary

<5>

<0>

<0>

<5>

<0>

<0>

Cyst, anterior lobe

1+

1

 

 

0

 

 

Cyst-like lesion, anterior lobe

1+

0

 

 

1

 

 

Thyroid

<5>

<0>

<0>

<5>

<0>

<0>

Ectopic thymic tissue

1+

0

 

 

1

 

 

Ultimobrancheal remnant

1+

1

 

 

1

 

 

Parathyroid

<5>

<0>

<0>

<5>

<0>

<0>

Adrenal

<5>

<0>

<0>

<5>

<0>

<0>

Brain

<5>

<0>

<0>

<5>

<0>

<0>

Spinal cord

<5>

<0>

<0>

<5>

<0>

<0>

Sciatic nerve

<5>

<0>

<0>

<5>

<0>

<0>

< > : Number of animals examined.

Grade; 1+ : Minimal, 2+ : Mild, 3+ : Moderate, 4+ : Severe,

Significantly different from 0 mg/kg group; * p <0.05, ** p <0.01

Sex

Organ Finding

Dose level (mg/kg)

Number of animals

Administration period

Recovery period

Non-pregnant

0

40

200

1000

0

1000

1000

12

11

12

11

5

5

1

Female                                  (Grade)

 Heart

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Lymph node, mandibular

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Lymph node, mesenteric

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Thymus

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Cyst

1+

3

 

 

1

 

 

 

Spleen

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Prostate

<5>

<0>

<0>

<5>

<0>

<0>

 

Extramedullary hematopoiesis, erythrocytic

1+

2

 

 

2

 

 

 

Bone marrow, femur

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Trachea

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Lung (and bronchus)

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Accumulation, foam cell, alveolus

1+

1

 

 

1

 

 

 

Mineralization, arterial wall, focal

1+

1

 

 

1

 

 

 

Stomach

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Cyst, mucosa, glandular stomach

1+

0

1

0

0

0

0

 

Hyperplasia, foveola, glandular stomach

1+

0

0

1

3

0

0

 

Necrosis, mucosa, glandular stomach, focal

1+

0

0

1

3

0

0

 

Small intestine, duodenum

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Small intestine, jejunum

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Small intestine, ileum

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Large intestine, cecum

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Large intestine, colon

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Large intestine, rectum

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Liver

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Proliferation, bile duct

1+

1

 

 

0

 

 

 

Kidney

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Basophilic tubule

1+

0

 

 

1

 

 

 

Cell infiltration, inflammatory, pelvis, focal

1+

0

 

 

1

 

 

 

Cell infiltration, lymphocyte, interstitium, focal

1+

1

 

 

0

 

 

 

Fibrosis, focal

1+

1

 

 

0

 

 

 

Urinary bladder

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Ovary

<5>

<0>

<0>

<5>

<0>

<0>

<1>

Uterus

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Vagina

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Pituitary

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Cyst, Rathke's pouch

1+

0

 

 

1

 

 

 

Thyroid

<5>

<0>

<0>

<5>

<0>

<0>

<0>

Ultimobrancheal remnant

1+

3

 

 

2

 

 

 

< > : Number of animals examined.

Grade; 1+ : Minimal, 2+ : Mild, 3+ : Moderate, 4+ : Severe,

Significantly different from 0 mg/kg group; * p <0.05, ** p <0.01

Fertility and pregnancy data in rats treated orally with the test chemical in the combined repeated dose and reproductive/developmental toxicity screening test

Dose level(mg/kg)

Administration period

0

40

200

1000

Number of pairs examined

12

12

12

12

Estrous cycle

4.18 ± 0.34

4.03 ± 0.09

4.00 ± 0.00

4.08 ± 0.29

Irregular estrous cycle

1/12

1/12

1/12

1/12

Number of pairs with successful mating

12

12

12

12

Mating index (%)a)

100.0

100.0

100.0

100.0

Number of pregnant females

12

12

12

12

Fertility index (%)b)

100.0

100.0

100.0

91.7

Pairing days until mating

3.0 ± 1.3

3.4 ± 1.8

2.8 ± 1.1

2.8 ± 1.1

Number of estrous stages without mating

0.0 ± 0.0

0.3 ± 0.5*

0.0 ± 0.0

0.0 ± 0.0

a) Mating index (%) = (Number of pairs with successful mating/number of pairs examined)×100

b) Fertility index (%) = (Number of pregnant animals/number of pairs with successful mating)×100

Values are expressed as Mean ± S.D.

Significantly different from 0 mg/kg group; * p <0.05, ** p <0.01

Delivery and litter data in rats treated orally with the test chemical in the combined repeated dose and reproductive/developmental toxicity screening test

Dose level(mg/kg)

Administration period

0

40

200

1000

Number of females examined

12

11

12

11

Number of females with live pups

12

11

12

11

Gestation index (%)a)

100.0

100.0

100.0

100.0

Gestation length (days)

22.4 ± 0.5

22.4 ± 0.5

22.3 ± 0.5

22.3 ± 0.5

Number of corpora lutea

16.3 ± 1.4

16.5 ± 1.8

17.3 ± 1.2

15.8 ± 1.9

Number of implantation sites

15.2 ± 1.4

15.2 ± 1.5

15.9 ± 1.0

14.8 ± 1.9

Implantation index (%)b)

92.94 ± 4.90

92.47 ± 5.65

91.98 ± 5.40

93.83 ± 6.46

Delivery index (%)c)

97.25 ± 3.41

94.67 ± 7.56

91.88 ± 10.99

92.85 ± 7.84

Number of pups delivered

14.8 ± 1.5

14.4 ± 1.7

14.6 ± 1.7

13.8 ± 2.6

Number of live pups on day 0

14.6 ± 1.6

14.4 ± 1.7

14.6 ± 1.7

13.6 ± 2.7

Number of live pups on day 4

14.5 ± 1.7

13.1 ± 3.1

14.5 ± 1.8

13.2 ± 2.6

Live birth index (%)d)

98.84 ± 2.72

100.00 ± 0.00

100.00 ± 0.00

98.55 ± 3.24

Viability index on day 4 (%)e)

99.31 ± 2.40

91.58 ± 19.79

99.36 ± 2.22

96.68 ± 4.03

Sex ratio of total number of offspring at birth (M/Total)

0.46(81/177)

0.50(79/158)

0.49(85/175)

0.45(68/152)

Sex ratio of total number of live

offspring at birth (M/Total)

0.46(80/175)

0.50(79/158)

0.49(85/175)

0.44(66/150)

Sex ratio of total number of live

offspring on day 4 (M/Total)

0.46(80/174)

0.51(73/144)

0.48(84/174)

0.44(64/145)

Sex ratio of total number of offspring at birth (M/Total, litter)

0.457 ± 0.112

0.505 ± 0.141

0.505 ± 0.141

0.453 ± 0.153

Sex ratio of total number of liveoffspring at birth (M/Total, litter)

0.456 ± 0.120

0.505 ± 0.141

0.484 ± 0.093

0.444 ± 0.157

Sex ratio of total number of live offspring on day 4 (M/Total, litter )

0.458 ± 0.117

0.506 ± 0.142

0.481 ± 0.094

0.444 ± 0.146

Body weight of pups (g)

on day 0 male

female

 

on day 4 male

female

6.9 ± 0.6

6.4 ± 0.5

 

10.7 ± 1.4

10.2 ± 1.3

6.6 ± 0.7

6.3 ± 0.8

 

10.4 ± 1.6

9.9 ± 1.5

6.8 ± 0.5

6.5 ± 0.6

 

10.2 ± 1.1

9.8 ± 1.1

6.8 ± 0.5

6.4 ± 0.5

 

10.7 ± 1.4

10.3 ± 1.4

a) Gestation index (%) = (Number of females with live pups/number of pregnant females)×100

b) Implantation index (%) = (Number of implantation sites/number of corpora lutea)×100

c) Delivery index (%) = (Number of pups delivered/number of implantation sites)×100

d) Live birth index (%) = (Number of live pups on day 0/number of pups delivered)×100

e) Viability index (%) = (Number of live pups on day 4/number of live pups on day 0)×100

Values are expressed as Mean±S.D.

Significantly different from 0 mg/kg group; * p <0.05, ** p <0.01

 

 

Conclusions:
Study 2: The NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested,while LOAEL for offspring in F3a generation was considered at 5mg/kg bw/day.When male and female Fischer 344 rats were treated with the test chemical orally.

Study 3: The NOAEL was considered to be 40 mg/kg/day for P and F1 generation when Crl:CD (SD) male and female rats treated with the test chemical orally by gavage.

Study 4: NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Crl:CD (SD) male and female rats treated with the test chemical orally by gavage.

Study 5: The No Observed Adverse Effect Level (NOAEL) of the test chemical in female rats was found at dose concentration of 10 mg/kg/day.
Executive summary:

Reproductive Toxicity Study:

The following summaries represent the information of the effect of the test chemical in the test animals:

Reproductive Toxicity Study 2:

In three generation reproductive toxicity study, Total 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group.90 animals of each sex were used in control group. The test chemical was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation. In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2ageneration. Same procedure was used to produce F2b generation. After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues. The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and lower dose group. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group. Also No dose related effects were observed on gross deformities in each generation. The only significant histopathological changes noted in F3ageneration were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female).Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen (p<0.001 for male and p<0.00001 for female). Hence the NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested. While LOAEL for offspring in F3a generation was considered at 5mg/kg bw/day when male and female Fischer 344 rats were treated with the test chemical orally.

Reproductive Toxicity Study 3:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD)male and female rats treated with the test chemical in the concentration of 0, 1.6, 8 and 40 mg/kg/day orally by gavage in 0.5 % Methylcellulose aqueous solution. 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity at 40 mg/kg bw. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female surviving rats at 40 mg/kg/day. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.Decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats and decrease in food consumption were observed in male and female rats as compared to control at40 mg/kg/day. Bradypnea, Prone position, Decrease in spontaneous activity,incomplete eyelid opening and abnormal gait in died animals and noeffects on surviving animals wereobserved.No effect on ureinanalysis was observed in male rats as compared to control. Similarly,Increase in the PLT was observed in male and female rats as compared to control at 40 mg/kg/day. Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat at 40 mg/kg/day. Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals. No effect on organ weight of treated was male and female rats were observed as compared to control. Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extremely killed animals and Light violet aqueous content in alimentary tract in surviving male and female rats at 40 mg/kg/day. Light violet aqueous content in stomach and cecum were observed in male rats (3/12) at 8 mg/kg/day. Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals and Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7), Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals at 40 mg/kg/day. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, development and growth of pups were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 40 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated with the test chemical orally by gavage.

Reproductive Toxicity Study 4:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD) male and female rats treated withthe test chemical in the concentration of 0, 40, 200 and 1000 mg/kg/day orally by gavage in 0.1 w/v% Tween 80 added 0.5 w/v% CMC-Na solution. No efect on survival, clinical signs, body weight, food consumption, behavior test, hematology, blood chemistry, urinalysis, organ weight and gross pathology were observed in treated male and female rats as compared to control. Focal necrosis of mucosa and hyperplasia of gastric pits in the glandular stomach were observed in female rat at 200 and 1000 mg/kg bw. The focal necrosis of mucosa is occasionally seen in the mucosa of the glandular stomach when the chemical compounds are administrated. Many of these induced changes have reversibility. Focal necrosis of mucosa were not noted at the end of the recovery period, indicating reversibility. The hyperplasia of mucosal epithelium in the glandular stomach was considered to be a regeneration or restoration image against early epithelium disorder. The image of hyperplasia disappeared by complete repairing at the end of the recovery period. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, clinical signs, body weights, development and growth of pups and gross pathological changes were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation whenCrl:CD (SD) male and female rats treated with the test chemical orally by gavage.

Reproductive Toxicity Study 5:

A study of the test chemical was performed to evaluate and assess the reproductive and developmental Toxicity of the test chemical in Sprague-Dawley Rats. In this evaluation, the maternal and embryo/fetal effects of

the test chemical orally administered to Sprague–Dawley rats once daily from day 6–15 of gestation at dose levels of 0, 10, 80, and 160 mg/kg/day. At dose levels of 80 and 160 mg/kg/day, the test chemical induced maternal toxicity, which consisted of severe reduction in maternal weight and food consumption during the treatment period. Doses of 80 and 160 mg/kg/day induced fetal skeletal abnormalities. The major skeletal defects were bipartite ossification of the thoracic centrum. The test chemical at 160 mg/kg/day also induced marked embryo lethality and visceral abnormalities. Based on the results of this study, a dose level of 10 mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for maternal and fetal developmental toxicity in rats.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Data is 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:

The following summaries represent the information of the effect of the test chemical in the test animals:

Reproductive Toxicity Study 2:

In three generation reproductive toxicity study, Total 450 male and female Fischer 344 rats were used in the study separated in two group i.e. control group and treated group.90 animals of each sex were used in control group. The test chemical was given in feed at dose concentration 0, 5, 15 and 30 mg /kg bw /day.45 animals of each sex were used for each dose concentration in F0 generation. In F0 generation male and female rats of same dose group were caged together for 14 days for mating .Pups from this mating F1a generation used for separate study while F0 generation animals were mating second times in same dose group following birth of F1b generation. At 100-140 days of age, one male and one female from each litter were randomly selected to produce F2ageneration. Same procedure was used to produce F2b generation. After 100-140 days, F3a generation were produce as result of mating one male and one female animal per litter from F2b generation. Pups from all generations were examined for gross deformities while at weaning, 2male and 2 female per litter of F3a generation were randomly selected for histopathology of organs and tissues. The dose related effect in body weight was observed at dose 30mg/kg bw /day dose group, as animals in this group had lower body weight compared to control and lower dose group. No dose related effects were observed on reproductive performance as number of pups per litter, fertility index and numbers of stillborn animals were not affected across the generation or dose group. Also No dose related effects were observed on gross deformities in each generation. The only significant histopathological changes noted in F3ageneration were dose related trend for focal dilation of renal cortex and tubules and statistically significant dose related trend for necrosis of thymus (p<0.012 for male and p<0.0001 for female).Also statistically significant inverse dose response relationship for red pulp hematopoietic cell proliferation of spleen (p<0.001 for male and p<0.00001 for female). Hence the NOAEL for reproductive toxicity was 30mg/kg bw/day the highest dose tested. While LOAEL for offspring in F3a generation was considered at 5mg/kg bw/day when male and female Fischer 344 rats were treated with the test chemical orally.

Reproductive Toxicity Study 3:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD)male and female rats treated with the test chemical in the concentration of 0, 1.6, 8 and 40 mg/kg/day orally by gavage in 0.5 % Methylcellulose aqueous solution. 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity at 40 mg/kg bw. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female surviving rats at 40 mg/kg/day. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.Decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats and decrease in food consumption were observed in male and female rats as compared to control at40 mg/kg/day. Bradypnea, Prone position, Decrease in spontaneous activity,incomplete eyelid opening and abnormal gait in died animals and noeffects on surviving animals wereobserved.No effect on ureinanalysis was observed in male rats as compared to control. Similarly,Increase in the PLT was observed in male and female rats as compared to control at 40 mg/kg/day. Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat at 40 mg/kg/day. Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals. No effect on organ weight of treated was male and female rats were observed as compared to control. Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extremely killed animals and Light violet aqueous content in alimentary tract in surviving male and female rats at 40 mg/kg/day. Light violet aqueous content in stomach and cecum were observed in male rats (3/12) at 8 mg/kg/day. Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals and Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7), Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals at 40 mg/kg/day. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, development and growth of pups were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 40 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated with the test chemical orally by gavage.

Reproductive Toxicity Study 4:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD) male and female rats treated withthe test chemical in the concentration of 0, 40, 200 and 1000 mg/kg/day orally by gavage in 0.1 w/v% Tween 80 added 0.5 w/v% CMC-Na solution. No efect on survival, clinical signs, body weight, food consumption, behavior test, hematology, blood chemistry, urinalysis, organ weight and gross pathology were observed in treated male and female rats as compared to control. Focal necrosis of mucosa and hyperplasia of gastric pits in the glandular stomach were observed in female rat at 200 and 1000 mg/kg bw. The focal necrosis of mucosa is occasionally seen in the mucosa of the glandular stomach when the chemical compounds are administrated. Many of these induced changes have reversibility. Focal necrosis of mucosa were not noted at the end of the recovery period, indicating reversibility. The hyperplasia of mucosal epithelium in the glandular stomach was considered to be a regeneration or restoration image against early epithelium disorder. The image of hyperplasia disappeared by complete repairing at the end of the recovery period. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, clinical signs, body weights, development and growth of pups and gross pathological changes were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation whenCrl:CD (SD) male and female rats treated with the test chemical orally by gavage.

Reproductive Toxicity Study 5:

A study of the test chemical was performed to evaluate and assess the reproductive and developmental Toxicity of the test chemical in Sprague-Dawley Rats. In this evaluation, the maternal and embryo/fetal effects of

the test chemical orally administered to Sprague–Dawley rats once daily from day 6–15 of gestation at dose levels of 0, 10, 80, and 160 mg/kg/day. At dose levels of 80 and 160 mg/kg/day, the test chemical induced maternal toxicity, which consisted of severe reduction in maternal weight and food consumption during the treatment period. Doses of 80 and 160 mg/kg/day induced fetal skeletal abnormalities. The major skeletal defects were bipartite ossification of the thoracic centrum. The test chemical at 160 mg/kg/day also induced marked embryo lethality and visceral abnormalities. Based on the results of this study, a dose level of 10 mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for maternal and fetal developmental toxicity in rats.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study 2:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD)male and female rats treated with the test chemical in the concentration of 0, 1.6, 8 and 40 mg/kg/day orally by gavage in 0.5 % Methylcellulose aqueous solution. 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity at 40 mg/kg bw. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female surviving rats at 40 mg/kg/day. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.Decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats and decrease in food consumption were observed in male and female rats as compared to control at40 mg/kg/day. Bradypnea, Prone position, Decrease in spontaneous activity,incomplete eyelid opening and abnormal gait in died animals and noeffects on surviving animals wereobserved.No effect on ureinanalysis was observed in male rats as compared to control. Similarly,Increase in the PLT was observed in male and female rats as compared to control at 40 mg/kg/day. Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat at 40 mg/kg/day. Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals. No effect on organ weight of treated was male and female rats were observed as compared to control. Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extremely killed animals and Light violet aqueous content in alimentary tract in surviving male and female rats at 40 mg/kg/day. Light violet aqueous content in stomach and cecum were observed in male rats (3/12) at 8 mg/kg/day. Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals and Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7), Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals at 40 mg/kg/day. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, development and growth of pups were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 40 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated with the test chemical orally by gavage.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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 data based on information of various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
other: OECD 422: Combined Repeated and Reproduction/Developmental Toxicity Screening Test
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The following experiments were performed to evaluate and assess the effect of the test chemical on developmental parameters in rats.
GLP compliance:
yes
Specific details on test material used for the study:
- Molecular weight :375.51 g/mol
- Substance type:Organic
Species:
other: Study 2 to 4: Rat
Strain:
other: Study 2 and 3: Crj:Cd (SD); Study 4: Sprague-Dawley
Details on test animals and environmental conditions:
Study 2: No Data Available
Study 3:
Study 4: After 2 weeks of acclimatization, Sprague–Dawley healthy virgin female rats(11–12 weeks old) (Guangzhou,China) were mated with males (13–14 weeks old) of the same rat strain from the same source in the late afternoon. Vaginal smears were taken the following morning until a positive identification of mating was made. The appearance of sperm in a vaginal smear or the presence of a vaginal plug was designated day 0 of gestation (GD 0). Sperm positive females were assigned to each study group based on body weight. The distribution of males to females was uniform among groups.The animals were individually housed in suspended stainless steel group cages,each with a wire mesh floor and front. The temperature in the animal room was maintained at 21–26 C, and relative humidity was 40–70%, with 12 air changes per hour and a 12 h light–dark cycle. Food and tap water were available ad libitum.
Route of administration:
other: Study 2 and 3 and 4: oral:gavage
Vehicle:
other: Study 2: 0.5 w/v%methylcellulose(MC)solution; Study 3: 0.1 w/v% Tween 80 added 0.5 w/v% CMC-Na solution Study 4: Tween-80
Details on exposure:
Study 2: PREPARATION OF DOSING SOLUTIONS: Dose were prepared by suspending test chemical in 0.5 % Methylcellulose aqueous solution at 0, 1.6, 8 and 40 mg/kg bw.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 % Methylcellulose aqueous solution
- Concentration in vehicle: 0, 1.6, 8 and 40 mg/kg bw.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required):
- Purity: No Data available

Study 3: PREPARATION OF DOSING SOLUTIONS: Test chemical suspended in 0.5 w/v% CMC-Na solution containing 0.1 w/v% Tween 80 at 0, 40, 200 and 1000 mg/kg bw

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 w/v% CMC-Na solution containing 0.1 w/v% Tween 80
- Concentration in vehicle: 0, 40, 200 and 1000 mg/kg bw
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Study 4: The test chemical suspensions at 1, 8, and 16 mg/mL were prepared in 1% Tween-80 (v/v, distilled water) within 6 h prior to each dosing.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Study 2: No data available
Study 3: No Data Available
Study 4: No Data Available
Details on mating procedure:
Study 2: No Data Available

Study 3: No Data Available

Study 4: After 2 weeks of acclimatization, Sprague–Dawley healthy virgin female rats(11–12 weeks old) from (Guangzhou,China) were mated with males (13–14 weeks old) of the same rat strain from the same source in the late afternoon. Vaginal smears were taken the following morning until a positive identification of mating was made. The appearance of sperm in a vaginal smear or the presence of a vaginal plug was designated day 0 of gestation (GD0). Sperm positive females were assigned to each study group based on body weight. The distribution of males to females was uniform among groups.
Duration of treatment / exposure:
Study 2: Male: 42 day
Female: 41 - 48 days (from 14 days before mating to day 4 of lactation)

Study 3: Males, 42 days
Females, from 14 days before mating to day 4 of lactation
Females (satellite), 42 days

Study 4: Daily from GD 6-15 of Gestation
Frequency of treatment:
Study 2: Daily

Study 3: Daily

Study 4: Once daily from day 6-15 of gestation dosing volume was 10 mL/kg
Duration of test:
Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available
Remarks:
Study 2: Doses / Concentrations:
0 (control), 1.6, 8, and 40mg/kg/day.
Study 3: 0 (control), 40, 200 and 1000 mg/kg/day
Study 4: Doses / Concentrations:
0 (control), 10, 40, 120, and 200 mg/kg/day.
Basis:
No. of animals per sex per dose:
Study 2: Total: 106 animals
0 mg/kg/day: 7 male, 12 female
1.6 mg/kg/day: 12 male, 12 female
8 mg/kg/day: 12 male, 12 female
40 mg/kg/day: 7 male, 12 female
Recory group:
0 mg/kg/day: 5 male, 5 female
40 mg/kg/day: 5 male, 5 female

Study 3: Total: 106
0 mg/kg/day: 7 male, 12 female
40 mg/kg/day: 12 male, 12 female
200 mg/kg/day: 12 male, 12 female
1000 mg/kg/day: 7 male, 12 female
Recory group:
0 mg/kg/day: 5 male, 5 female
1000 mg/kg/day: 5 male, 5 female

Study 4: Eight sperm positive females/group
Control animals:
yes, concurrent vehicle
Details on study design:
Study 2: No Data Available
Study 3: No Data Available
Study 4: To determine the dose range, eight sperm positive females/group were given test chemical by gavage daily from GD 6–15 at dosages of 0 (control), 10, 40, 120, and 200 mg/kg/day. The results show that the administration of 200 mg/kg/day caused maternal death (2 of 8 treated pregnant rats). The post-implantation loss was found at 120–200 mg/kg/day. Minor changes of body weight were revealed at 40 mg/kg/day. No evidence of adverse effects was found at 10 mg/kg/day.Based on these results, 30 sperm positive females/group were administered with the test chemical by gavage at 0 (control), 10, 80, and 160 mg/kg/day. Dosing occurred once daily in the morning from GD 6–15. The dosing volume was 10 mL/kg. Initial doses were based on GD 6 weight and adjusted every 3 days throughout the treatment period. A control group received the vehicle under the same conditions. Caesarean sections were performed on GD 20, and fetuses were examined for developmental effects.
Maternal examinations:
Study 2: Survival, clinical sign, body weight, food consumption and urineanalysis, FOB, Urinalysis (Male only), were examined. Estrous cyclicity, copulation and implantation were examined. Hematology, clinical chemistry, Organ weight, gross pathology and histopathology were examined.

Study 3: Survival, clinical sign, body weight, food consumption and urine analysis and FOB were examined. Estrous cyclicity, copulation and implantation were examined. Haematology, clinical chemistry, Organ weight, gross pathology and histopathology were examined.

Study 4: Animals were observed twice daily before and after treatment for signs of overt toxicity, changes in general appearance, and mortality. Sperm positive females were weighed on GD0, 3, 6, 9, 12, 15, and 20. Final GD20 body weights were corrected for gravid uterine weights. Food consumption was measured on GD0, 6, 9, 12, 15, and 19. Water consumption was monitored daily. Three females in each group were euthanized by deep anesthesia with sodium pentobarbital followed by exsanguination on GD7 and GD16, respectively. At least 2 mL blood was collected from each animal via the abdominal aorta under deep anesthesia. At least 1 mL amniotic fluid were collected from each dam euthanized on GD16. Blood samples were placed in iced water until they were centrifuged to prepare the plasma samples. All plasma and amniotic fluid samples were stored at 80 C until analysis. On GD20, the surviving mated females were euthanized by CO2 asphyxiation followed by exsanguination. The blood and amniotic fluid samples were collected from the first three pregnant animals necropsied in each group, as described above. All the study animals were examined for gross abnormalities. The livers of pregnant rats in the highest dose group and in the control group were fixed in 10% buffered formalin on GD20. A full histopathological examination was performed on hematoxylin- and eosin-stained tissue sections of the liver.
Ovaries and uterine content:
Study 2: Number of corpora lutea,number of implantations, implantation index live birth index, delivery index were examined.

Study 3: Estrous cycle, copulation index, fertility index, delivery index, gestation length, numbers of corpora lutea or implantations, implantation index, gestation index, parturition, or maternal behavior was examined

Study 4: After necropsy on GD20, the uteri were removed and weighed. The number of total implantations and corpora lutea, the number and location of viable and non-viable fetuses, early and late resorptions, and all externally visible fetal abnormalities were recorded
Fetal examinations:
Study 2: Number of pups, number of live pups, sex ratio on days 0 and 4, clinical signs and body weight were examined. Gross pathology was performed. Viability index on 0 and 4 day were examined.

Study 3: The number of offspring or live offspring, sex ratio, live birth index, or viability index on day 4 was examined. Also, results from external features, clinical signs, body weights, or necropsy of the offspring were determined.

Study 4: After necropsy on GD20, the uteri were removed and weighed. The number of total implantations and corpora lutea, the number and location of viable and non-viable fetuses, early and late resorptions, and all externally visible fetal abnormalities were recorded. All live fetuses and placentas were weighed. The crownrump length of all live fetuses was measured and their sex was determined. Uteri from females that appeared non-gravid and individual uterine horns without visible implantations were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. All fetuses were examined for external abnormalities. Approximately one-half of the fetuses in each litter were eviscerated (the livers and kidneys of these fetuses were weighed) and stained with Alcian blue and Alizarin red S for subsequent skeletal examination (Tyl and Marr, 2005). The remaining fetuses were fixed in Bouin’s solution for soft tissue examination according to Wilson’s method (Tyl and Marr, 2005). The descriptions of the variations and malformations were in accordance with the terms for developmental abnormalities in common laboratory animals
Statistics:
Study 2, 3 and 4: No Data Available
Indices:
Study 2: Pup Viability Index, Sex Ratio.
Study 3: Implantation Index, Resorption Index, Gestational Index, Viability Index were observed.
Study 4: Pup Viability Index, Implantation Index, Resorption Index
Historical control data:
Study2, 3 and 4: No Data Available
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female survival rats were observed. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.

Study 3: No clinical signs were observed in treated rats as compared to control.

Study 4: Perineal yellowish to brownish staining was noted in two high-dose females on GD11 and GD12, respectively. There were reddish substances
on the tray under the two animals’ cages. Additionally, Alopecia was observed in seven females at 160 mg/kg/day, and it was first noted on GD9 or later. No other treatment-related clinical findings were noted.
Dermal irritation (if dermal study):
not specified
Mortality:
mortality observed, treatment-related
Description (incidence):
Study 2: When treated with 40 mg/kg bw, 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control.

Study 3: No effect on survival of treated rats were observed at 40, 200 and 1000 mg/kg bw as compared to control.

Study 4: All females survived up to the scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats as compared to control.

Study 3: No changes were observed in animals at any dose levels.

Study 4: Average maternal body weight in each group before dosing (body weight of GD0, 3, 6) were similar, whereas body weights after dosing (GD9, 12, 15, and 20) in the 80 and 160 mg/kg/day dose group were significantly less than those in the control group.The body weight gain decreased in a dose-related manner during treatment period (GD6–15). Thus, the 80 mg/kg/day dose animals gained less than half the weight gain of the control group, while the 160 mg/kg/day dose rats lost weight. Weight gain was decreased, especially during the early period of treatment (GD 6–9).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, decrease in food consumption was observed in male and female rats as compared to control.
Study 3: No changes were observed in animals at any dose levels.
Study 4: During the period of administration (GD6–15), significant decrease in maternal food consumption was observed at 80 and 160 mg/kg/day.
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 2: When treated with 40 mg/kg/day, Increase in the PLT was observed in male and female rats as compared to control.
Study 3: No changes were observed in animals at any dose levels.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat.
Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and Increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals.

Study 3: No changes were observed in animals at any dose levels.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No changes were observed in animals at any dose levels.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, Bradypnea, Prone position, Decrease in spontaneous activity, Incomplete eyelid opening and abnormal gait in died animals and no effect on surviving animals were observed.

Study 3: No changes were observed in animals at any dose levels.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No changes were observed in animals at any dose levels.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extrimlly killed animals.

When treated with 40 mg/kg/day, Light violet aqueous content in alimentary tract in surviving male and female rats.

When treated with 8 mg/kg/day, Light violet aqueous content in stomach and cecum were observed in male rats (3/12) as compared to control.

Study 3: No changes were observed in animals at any dose levels.


Study 4: No gross abnormalities were found in the dams at necropsy.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Study 2: When treated with 40 mg/kg/day, Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals. Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7) and Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals.

Study 3: No Data Available

Study 4: Upon histopathological examination of the maternal liver lesions in the highest-dose group and in the controls, there were no abnormal observations.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Study 2, 3 and 4: No Data Available
Number of abortions:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No effects were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No effects were observed.

Study 4: The pre-implantation loss were not affected by the test chemical. The post-implantation loss was 59% at 160 mg/kg/day, whereas it was 8%, 7%, and 10% at 0, 10, and 80 mg/kg/day. At 160 mg/kg/day, most of the post-implantation loss (98%) consisted in resorption.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Study 2, 3 and 4: No effects were observed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No effects were observed.

Study 4: At 160 mg/kg/day, most of the post-implantation loss (98%) consisted in resorption. The incidence of dams with more than one resorption increased with the dose. In the 160 mg/kg/day dose group, 10 of 21 dams had completely resorbed litters, two of which were not visible macroscopically.
Dead fetuses:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No effects were observed.

Study 4: The death of fetuses was mostly observed in 160 mg/kg.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No effects were observed.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed.

Study 3: No effects were observed.
Other effects:
not specified
Details on maternal toxic effects:
Study 2: No Data Available

Study 3: No effects were observed on maternal parameters at any given dose levels.

Study 4: Details on maternal toxic effects:
At dose levels of 80 and 160 mg/kg/day, The test chemical induced maternal toxicity, which consisted of severe reduction in maternal weight and food consumption during the treatment period. Based on the results of this study, a dose level of 10mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in rats.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
gross pathology
maternal abnormalities
number of abortions
pre and post implantation loss
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
necropsy findings
Remarks on result:
other: Not Specified
Abnormalities:
not specified
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Study 4: The test chemical induced a dose-related reduction in fetal body weight and placental weight which reached the statistical significance at 160 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Study 4: The most reductions in number of live offsprings were observed in the higher dose of 160 mg/kg.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Study 4: No effects were observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
External malformations:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Study 4: The incidences of fetuses and litters with external abnormality in the treated groups were not significantly different from those in the control group. A few cases (1 or 2 per group) of anasarca, exencephaly, and hindlimb hyperextension were only observed at 80 and 160 mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Study 4: The test chemical induced a dose-related increased incidence of fetuses (litters) with skeletal abnormalities. The number of affected fetuses (litters) was statistically significant in the two higher-dose groups compared with the control. The predominant skeletal anomalies observed were bipartite ossification of the thoracic centrum. The fetal(litter)incidences of bipartite ossification of the thoracic centrum were significantly higher than control at 80 and 160 mg/kg/day, and were 33% (74%) and 69% (100%), respectively. At 160 mg/kg/day, the etal and litter incidences of bipartite ossification of the lumbar centrum were 8% and 36%, also significantly higher than that of the control, in which no fetus with this abnormality was observed. Other skeletal abnormalities, such as detached rib cartilage, supernumerary thoracic centrum, absent lumbar centrum, supernumerary lumbar centrum, and misshapen sacral arch, were only observed in the dose groups, but were not significantly increased.
Visceral malformations:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Study 4: Significantly increased incidences of fetuses and litters with visceral abnormality were detected only in the 160 mg/kg/day group compared with the control values. No specific visceral alteration was significantly different among the four groups. Some visceral abnormalities, such as misshapen cerebrum, enlarged ventricular chamber, small kidney, dilated ureter, small lung, and supernumerary lung lobe, were only observed in the treated groups.
Other effects:
no effects observed
Description (incidence and severity):
Study 2: No effects were observed
Study 3: No effects were observed
Details on embryotoxic / teratogenic effects:
Study 2, 3: No effects observed in any developmental Toxicity parameter.
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
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: The NOAEL was considered to be 40 mg/kg/day for P and F1 generation when Crl:CD (SD) male and female rats treated with the test chemical orally by gavage.
Study 3: NOAEL was considered to be 1000 mg/kg/day for P and F1 generation when Crl:CD (SD) male and female rats treated with the test chemical orally by gavage.
Study 4: The No Observed Adverse Effect Level (NOAEL) of the test chemical in female rats was found at dose concentration of 10 mg/kg/day.

Executive summary:

Developmental Toxicity Study:

The following summaries represent the data for the developmental toxicity data for the test chemical:

Developmental Toxicity Study 2:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD)male and female rats treated with the test chemical in the concentration of 0, 1.6, 8 and 40 mg/kg/day orally by gavage in 0.5 % Methylcellulose aqueous solution. 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity at 40 mg/kg bw. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female surviving rats at 40 mg/kg/day. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.Decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats and decrease in food consumption were observed in male and female rats as compared to control at40 mg/kg/day. Bradypnea, Prone position, Decrease in spontaneous activity,incomplete eyelid opening and abnormal gait in died animals and noeffects on surviving animals wereobserved.No effect on ureinanalysis was observed in male rats as compared to control. Similarly,Increase in the PLT was observed in male and female rats as compared to control at 40 mg/kg/day. Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat at 40 mg/kg/day. Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals. No effect on organ weight of treated was male and female rats were observed as compared to control. Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extremely killed animals and Light violet aqueous content in alimentary tract in surviving male and female rats at 40 mg/kg/day. Light violet aqueous content in stomach and cecum were observed in male rats (3/12) at 8 mg/kg/day. Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals and Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7), Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals at 40 mg/kg/day. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, development and growth of pups were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 40 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated with the test chemical orally by gavage.

Developmental Toxicity Study 3:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD) male and female rats treated withthe test chemical in the concentration of 0, 40, 200 and 1000 mg/kg/day orally by gavage in 0.1 w/v% Tween 80 added 0.5 w/v% CMC-Na solution. No efect on survival, clinical signs, body weight, food consumption, behavior test, hematology, blood chemistry, urinalysis, organ weight and gross pathology were observed in treated male and female rats as compared to control. Focal necrosis of mucosa and hyperplasia of gastric pits in the glandular stomach were observed in female rat at 200 and 1000 mg/kg bw. The focal necrosis of mucosa is occasionally seen in the mucosa of the glandular stomach when the chemical compounds are administrated. Many of these induced changes have reversibility. Focal necrosis of mucosa were not noted at the end of the recovery period, indicating reversibility. The hyperplasia of mucosal epithelium in the glandular stomach was considered to be a regeneration or restoration image against early epithelium disorder. The image of hyperplasia disappeared by complete repairing at the end of the recovery period. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, clinical signs, body weights, development and growth of pups and gross pathological changes were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation whenCrl:CD (SD) male and female rats treated with the test chemical orally by gavage.

Developmental Toxicity Study 4:

A study of the test chemical was performed to evaluate and assess the reproductive and developmental Toxicity of the test chemical in Sprague-Dawley Rats. In this evaluation, the maternal and embryo/fetal effects of

the test chemical orally administered to Sprague–Dawley rats once daily from day 6–15 of gestation at dose levels of 0, 10, 80, and 160 mg/kg/day. At dose levels of 80 and 160 mg/kg/day, the test chemical induced maternal toxicity, which consisted of severe reduction in maternal weight and food consumption during the treatment period. Doses of 80 and 160 mg/kg/day induced fetal skeletal abnormalities. The major skeletal defects were bipartite ossification of the thoracic centrum. The test chemical at 160 mg/kg/day also induced marked embryo lethality and visceral abnormalities. Based on the results of this study, a dose level of 10 mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for maternal and fetal developmental toxicity in rats.
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:
Data is 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 following summaries represent the data for the developmental toxicity data for the test chemical:

Developmental Toxicity Study 2:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD)male and female rats treated with the test chemical in the concentration of 0, 1.6, 8 and 40 mg/kg/day orally by gavage in 0.5 % Methylcellulose aqueous solution. 1 male and 4 female died and 3 male and 1 female were sacrificed due to morbidity at 40 mg/kg bw. No effect on survival of treated rats were observed at 1.6 and 8 mg/kg bw as compared to control, Decreased spontaneous activity, Prone position, Bradypnea, Abnormal respiratory tones, Hypothermia, Abnormal gait, Soft stool, Emaciation, Abdominal distention, Dirty around anus, Soiled perineal region, External genital bleeding and test article-colored feces were observed in dead treated rats as compared to control. Soft stool and dirty around anus in male and female and External genital bleeding in Female surviving rats at 40 mg/kg/day. Colored feces were observed in 1.6 and 8 mg/kg bw treated rats.Decrease in body-weight and body-weight gain were observed in male and body weight gain in female rats and decrease in food consumption were observed in male and female rats as compared to control at40 mg/kg/day. Bradypnea, Prone position, Decrease in spontaneous activity,incomplete eyelid opening and abnormal gait in died animals and noeffects on surviving animals wereobserved.No effect on ureinanalysis was observed in male rats as compared to control. Similarly,Increase in the PLT was observed in male and female rats as compared to control at 40 mg/kg/day. Increase in the AST and ALT in male, CPK and BUN were observed in extremely killed male and female rat at 40 mg/kg/day. Decrease in the TP and alpha 1-glb, Increase in the Alb, A/G and BUN in male and increase in the BUN (tendency) in Female rats were observed as compared to control in survival animals. No effect on organ weight of treated was male and female rats were observed as compared to control. Light violet aqueous content and discoloration of mucus membrane in all of the alimentary tract containing oral cavity, subcutaneous tissues and uterus (sporadically noticed in Male and Female), Hydrothorax in thoracic cavity (Male 1/4, Female 2/5), Small thymus (Male 3/4, Female 2/5), Small spleen (Male 1/4, Female 2/5), Edema in thymus (Male 1/4), Reddish urine in gallbladder (Male 1/4), Red discoloration in mucosa of the bladder (Male 1/4), Red discoloration of testis (Male 1/4), Red discoloration of adipose tissue around the testis (Male 1/4), Dilatation of stomach (Female 4/5), Enlargement of adrenal (Female 4/5), Gas retention in stomach (Female 2/5), Dark red viscous retention in vagina (Female 2/5), Dilatation of cecum (Female 1/5), Gas retention in cecum (Female 1/5) observed in extremely killed animals and Light violet aqueous content in alimentary tract in surviving male and female rats at 40 mg/kg/day. Light violet aqueous content in stomach and cecum were observed in male rats (3/12) at 8 mg/kg/day. Trachea; Desquamation of epithelium and inflammatory cell infiltration of mucosa (Male 1/4, Female 2/5), Glandular stomach; Atrophy of epithelial cell (Male 1/4, Female 1/5), Small/large intestine; Hypertrophy of epithelial cell (sporadically observed in Male and Female), Liver; Hypertrophy of centrilobular hepatocyte (Male 3/4, Female 3/5), Necrosis (Male 1/4, Female 2/5), Vacuolation (Male 1/4), Adrenal; Hypertrophy of zona fasciculata (Male 2/4, Female 5/5), Bone marrow; Deficient erythropoiesis and granulopoiesis (Male 3/4, Female 2/5), Spleen; Atrophy of follicle / marginal zone (Male 2/4, Female 5/5) and periarterial lymphatic sheath (Male 3/4, Female 5/5), Thymus; Atrophy (Male 4/4, Female 3/5) /necrosis of lymphocyte (Male 3/4, Female 4/5), Lymph node; Atrophy of follicle / paracortex (Male 3/4, Female 5/5), Spinal cord / fourth ventricle / testis / urinary bladder; Hemorrhage or hemorrhagic infarction (Male 1/4), Vagina; Hemorrhage (Female 2/5), Mucoid degeneration of mucosa (Female 2/5), Lung; Hemorrhage of alveolus, edema of alveolus and inflammatory cell infiltration (Female 1/5) were observed as compared to control in extremis killed animals and Liver; Hypertrophy of centrilobular hepatocyte (Male 2/4), Duodenum; Hypertrophy of epithelial cell (Male 2/4, Female 6/7), Mesenteric lymph node and Sinus histiocytosis (Male 1/4, Female 3/7) were observed as compared to control in survival animals at 40 mg/kg/day. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, development and growth of pups were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 40 mg/kg/day for P and F1 generation whenCrl:CD (SD)male and female rats treated with the test chemical orally by gavage.

Developmental Toxicity Study 3:

In a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test,Crl:CD (SD) male and female rats treated withthe test chemical in the concentration of 0, 40, 200 and 1000 mg/kg/day orally by gavage in 0.1 w/v% Tween 80 added 0.5 w/v% CMC-Na solution. No efect on survival, clinical signs, body weight, food consumption, behavior test, hematology, blood chemistry, urinalysis, organ weight and gross pathology were observed in treated male and female rats as compared to control. Focal necrosis of mucosa and hyperplasia of gastric pits in the glandular stomach were observed in female rat at 200 and 1000 mg/kg bw. The focal necrosis of mucosa is occasionally seen in the mucosa of the glandular stomach when the chemical compounds are administrated. Many of these induced changes have reversibility. Focal necrosis of mucosa were not noted at the end of the recovery period, indicating reversibility. The hyperplasia of mucosal epithelium in the glandular stomach was considered to be a regeneration or restoration image against early epithelium disorder. The image of hyperplasia disappeared by complete repairing at the end of the recovery period. In addition no reproductive effect such as Estrous cyclicity, copulation and implantation, Fertility rat, gestation period, implantation index, live birth index, delivery index were observed in treated rats as compared to control. No effect on viability, clinical signs, body weights, development and growth of pups and gross pathological changes were observed in treated rats as compared to control. Therefore, NOAEL was considered to be 1000 mg/kg/day for P and F1 generation whenCrl:CD (SD) male and female rats treated with the test chemical orally by gavage.

Developmental Toxicity Study 4:

A study of the test chemical was performed to evaluate and assess the reproductive and developmental Toxicity of the test chemical in Sprague-Dawley Rats. In this evaluation, the maternal and embryo/fetal effects of

the test chemical orally administered to Sprague–Dawley rats once daily from day 6–15 of gestation at dose levels of 0, 10, 80, and 160 mg/kg/day. At dose levels of 80 and 160 mg/kg/day, the test chemical induced maternal toxicity, which consisted of severe reduction in maternal weight and food consumption during the treatment period. Doses of 80 and 160 mg/kg/day induced fetal skeletal abnormalities. The major skeletal defects were bipartite ossification of the thoracic centrum. The test chemical at 160 mg/kg/day also induced marked embryo lethality and visceral abnormalities. Based on the results of this study, a dose level of 10 mg/kg/day is considered the no-observed-adverse-effect-level (NOAEL) for maternal and fetal developmental toxicity in rats.

Justification for classification or non-classification

From the NOAEL and LOAEL values on reproduction and developmental toxicities it is concluded that the test chemical may not be classified as a reproductive or developmental toxicant according to CLP regulation.