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Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422, GLP, Key, Reliability 2): NOEL = 10 mg/kg bw/day in rats.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD TG 422 (1996) with the following deviations compared to the current version (2016): - hematology and clinical biochemistry determinations were performed on male animals only and in the control and high dose groups only; - thyroid hormones were not assessed; - neurological assessments were not performed; - individual tables are not available; - historical control data are not available.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
yes
Remarks:
Hematology and clinical biochemistry determinations were performed on male animals only and in the control and high dose groups only
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in an airtight container at room temperature in a dark location with nitrogen replacement
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: confirmed to be stable for eight days when stored at room temperature in a dark location with nitrogen replacement.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The solution was prepared twice a week.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): none reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: 7 weeks (administration commenced at 8 weeks of age)
- Weight at study initiation: 294 to 319 g for males and 195 to 223 g for females
- Fasting period before study: no
- Housing: individually in a metal mesh cage (W190 × D350 × H170 mm: Lead Engineering Co., Ltd.) except in the mating period and the period from Day 17 of pregnancy to Day 4 of nursing. During the mating period, a total of two animals, consisting of one each of male and female animals, were kept in a metal mesh cage (W266 × D266 × H200 mm: Riko Denki K.K.); also, female animals were reared individually in a plastic Econ cage (W340 × D400 × H185 mm: Clea Japan, Inc.) containing wood chips (White Flake: Charles River Laboratories Japan, Inc.) from Day 17 of pregnancy to Day 4 of nursing.
- Diet (e.g. ad libitum): free access to solid feed (NMF: Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): free access to drinking water (Fujimi Waterworks Union)
- Acclimation period: One week

DETAILS OF FOOD AND WATER QUALITY:
For the analysis of the contaminants in the feed, the data from the analysis performed by Japan Food Research Laboratories were used; for the analysis of drinking water, periodic (four times per year) analysis of water quality according to the Water Supply Law was requested from the Shizuoka Life Science Testing Center, and the analysis data were obtained. These data were observed for any impact on the results of the study before being stored.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5°C ± 3.5°C
- Humidity (%): 50% ± 20%,
- Air changes (per hr): 10 to 15 times per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Not reported
Route of administration:
oral: gavage
Details on route of administration:
Oral administration was specified for this study with consideration for the possibility for the continued ingestion of the test substance in humans and in accordance with the OECD Guidelines for the Testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening test. With reference to these guidelines, the administration period was set as a total of 49 days for males, consisting of 14 days before mating and 35 subsequent days, and 41 to 46 days for females, consisting of 14 days before mating, mating period (up to 5 days), pregnancy period, and 4 days of nursing until before the day of necropsy.
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
One day’s amount of the prepared test solution was dispensed into each brown glass bottle then stored at room temperature in a dark location after performing nitrogen replacement.
The test substance was administered by gavage once daily from 9:00 to 16:30 using a metallic gastric tube.
The amount of the solution to be administered to each animal was calculated from the body weights on each day of measurement for males from the body weights on each day of measurement for females before and during mating, body weights on Days 0, 7, 14, and 21 of pregnancy for females during pregnancy, and body weights on Day 0 of nursing for females in the nursing period.

VEHICLE
- Justification for use and choice of vehicle: not reported
- Concentration in vehicle: 0.05, 0.2, 0.8 and 3.2 % (w/v). The test substance was not converted by purity but displayed as the weight of the technical product.
- Amount of vehicle (if gavage): 0.5 mL/100 g.
- Lot/batch no. (if required): 4912, 4×12
- Purity: not reported
- Source: Maruishi Pharmaceutical Co., Ltd
- Storage conditions: Store in an airtight container at room temperature with nitrogen replacement
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test solution was confirmed twice: before the start of administration for males and females and at the final week of administration for the males. As a result of HPLC performed at BoZo Research Center Inc. for the solutions of all concentrations, the percentage to the labeled value was within the range of 96.9% to 104.0% for all solutions, indicating appropriate concentrations
Duration of treatment / exposure:
Males: 49 days, from two weeks pre-mating and throughout the mating period to the day before necropsy.
Females: 41 to 46 days from two weeks pre-matin, throughout mating, gestation, up to Day 3 of lactation.
Frequency of treatment:
Once daily
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
160 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage was determined with reference to the results of the previously conducted two-week oral dose preliminary study in rats (BoZo Research Center Inc., study No.: R-559, dose: 0, 50, 100, 200, and 400 mg/kg).
- Rationale for animal assignment: computerized block placement method
- Fasting period before blood sampling for clinical biochemistry: fasted overnight (about 16 hours)
- Rationale for selecting satellite groups:not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Dose range finding studies: In the preliminary study study, all five males and four of five females in the 400 mg/kg group as well as one of five males and two of five females in the 200 mg/kg group died by Day 7 of administration. However, the test substance had no notable impact on the general condition, body weights, food consumption, hematology parameters, and blood biochemistry parameters in the 100 mg/kg and lower groups. Based on these results, the maximum dose for this study was set as 160 mg/kg, which is approximately intermediate of the 200 mg/kg where deaths were observed and 100 mg/kg where no impact of the test substance was observed. This dose was divided by the common ratio of 4 to set the doses of 40, 10, and 2.5 mg/kg.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times daily before administration, immediately after administration, and 2 hours after administration. However, the observations on holidays were conducted twice daily, before administration and immediately after administration, and the observations on the day of necropsy were conducted once prior to necropsy
- Cage side observations checked: symptoms of poisoning and behavioral abnormalities

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: measured from 9:00 to 10:00 on the day of commencing administration (before administration on Day 1) and Days 4, 8, 11, 15, 22, 29, 36, 43, and 50.

FOOD CONSUMPTION: Yes:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
For the measurement of food consumption, the residual amount from the previous day was measured from 9:30 to 10:00 on the same days as the measurement of body weights (excluding the day of commencing administration and the day of necropsy), and the daily food consumption was calculated from the difference to the amount of food provided.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Males only)
- Time schedule for collection of blood: At the time of sacrifice on the day after the completion of the administration period
- Anaesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: Yes (overnight - about 16 hours)
- How many animals: all males
- Parameters checked in table No.7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes (males only)
- Time schedule for collection of blood: At the time of sacrifice on the day after the completion of the administration period
- Animals fasted: Yes (overnight - about 16 hours)
- How many animals: all males
- Parameters checked in table No.7.5.1/1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.1/2)

HISTOPATHOLOGY: Yes (see table 7.5.1/2)
Optional endpoint(s):
Optional endpoints: No
Other examinations:
Observation of female sexual cycles, mating, delivery and nursing. Observation of neonates.
(cf. IUCLID section 7.8 - Toxicity to reproduction)
Statistics:
Statistical analysis was performed using the following test methods with the significance level of 5% and 1%.
(1) Multiple test
Tests on the equal variance in each group were performed by Bartlett’s method. If the test resulted in equal variance, one-way ANOVA was performed; if significant intergroup change was found, paired comparison test was performed by Dunnett’s method if the number of animals in each group was equal, and the Scheffé method if not equal. If the variance was not equal, Kruskal-Wallis’ ranked test was performed; if the result was significant, the difference between the mean rank between the control group and each treatment group were subject to testing by Dunnett’s method, if the number of animals in each group was equal and Scheffé method if not equal. Body weight, food consumption, number of estrus image obtained, sexual cycle, number of days of rearing together, pregnancy period, hematology parameters, blood biochemistry parameters, organ weights, number of corpora luteum, number of implantation sites, number of pups, implantation index, sex ratio, delivery index, live birth index, neonate survival rate
(2) χ2 test
Copulation index, fertility index, live birth index
Clinical signs:
no effects observed
Description (incidence and severity):
No changes were observed in the general condition throughout the study for any animals in the control group and the test substance groups.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Deaths occurred in one maternal animal in the 40 mg/kg group during delivery on Day 22 of pregnancy and one maternal animal in the 160 mg/kg on Day 22 of pregnancy. No abnormalities were observed in the general condition of the animal from 160 mg/kg that resulted in death until the discovery of death. The same animal was observed with the formation of nodules on the left lateral part of the neck on Day 11 of pregnancy, although this is not shown in the tables. There were two animals in the 160 mg/kg group (No. 5101, 5102) with papillary underdevelopment; all nursed pups of these animals had died by Day 2 or 3 of nursing, and these maternal animals were euthanized at this point.
In conclusion, no common findings considered to be directly related to death were observed in either animal as a result of histopathology performed on dead animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males: The test substance groups showed similar changes in body weight to the control group. No significant difference was observed in the body weights at each day of measurement and the changes in body weight gain during the administration period (Days 1 to 43) in comparison with the control group.
Females: The test substance groups showed similar changes in body weights to the control group throughout the pre-mating, pregnancy, and nursing periods. No significant difference was observed in the body weights at each day of measurement, and the changes in body weight gain, except for the significantly low value of body weight on Day 0 of nursing and significantly high value of body weight gain from Days 0 to 4 of nursing in the 2.5 mg/kg group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males: The test substance groups showed similar changes in food consumption to the control group; however, the values obtained on Day 36 in the 40 mg/kg group and on Days 15, 36, and 43 in the 160 mg/kg group were significantly higher than those of the control group.
Females: The test substance groups showed similar changes in food consumption to the control group throughout the pre-mating, pregnancy, and nursing periods. No significant difference was observed between the control group and the test substance groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: No difference was observed in any test parameters between the test substance groups at 40 mg/kg and lower and the control group. Meanwhile, high mean corpuscular volume, low mean corpuscular hemoglobin concentration, and prolonged prothrombin time were observed in the 160 mg/kg group with a significant difference compared with the control group. No significant difference was observed between the control group and the 160 mg/kg group for any other parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: No difference was observed in any test parameters between the test substance groups at 40 mg/kg and lower and the control group. Meanwhile, low chlorine, high A/G ratio and albumin ratio, and low γ-globulin ratio were observed in the 160 mg/kg group with a significant difference compared with the control group. No significant difference was observed between the control group and the 160 mg/kg group for any other parameters.
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males: No significant difference was found in absolute and relative weights between the control group and the test substance groups.
Females: No significant difference was observed in absolute and relative weights between the test substance groups at 40 mg/kg and lower and the control group. In the 160 mg/kg group, significantly higher liver weight and significantly low right ovary weight were observed, both in absolute and relative weights. No significant difference was observed for the absolute and relative weights of any other organs in this group in comparison with the control group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males: No abnormality was found in the any animals of the control group and the test substance groups with regard to necropsy findings.
Females: The animal that died from the 40 mg/kg group (No. 4102) showed thymus atrophy, partial discoloration of the liver, and enlargement of adrenal gland. Also, the animal that died from the 160 mg/kg group (No. 5110) showed subcutaneous nodules on the left lateral part of the neck, dark red punctate scattering in the thymus, and dark red changes in the lungs. The maternal animals with all pups dead (No. 5101, 5102) showed papillary underdevelopment as external observations. No abnormalities were observed in the necropsy findings of all other animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males: Changes thought to be caused by the administration of the test substance were observed in the liver. The extent and frequency of the centrilobular fatty changes in the liver were similar to the control group for the 2.5 and 10 mg/kg groups, while these tended to decrease in the 40 mg/kg group and decreased in the 160 mg/kg group. Because of the observation of vacuoles in the hepatocyte cytoplasm in the peripheral region of the hepatic lobule, Oil Red O staining was performed for representative animals from the control group, 40 mg/kg group, and 160 mg/kg group. As a result of this investigation, a positive result was observed for Oil Red O staining according to the extent of vacuole in the hepatocyte cytoplasm, which led to identification of these vacuoles as fats. In addition, slight localized myocarditis was observed in the hearts of two animals from the control group (1006, 1011) and four animals from the 160 mg/kg group (5002, 5003, 5004, 5009), mild localized basophilization of the tubular epithelium was observed in the kidneys of one animal from the control group (1007), and slight localized seminiferous tubule atrophy was observed in the testes of one animal from the control group (1004).
Females: Findings in the animals who died and the animals euthanized because of the deaths of all pups were as follows: The animal that died from the 40 mg/kg group (4102) showed a moderate centrilobular necrosis in the liver, mild enlargement of fascicular zone in the adrenal gland, and mild atrophy of the thymus; animal that died from the 160 mg/kg group (5110) showed a moderate myocardial degeneration in the heart, mild tubular epithelial cell necrosis in the kidneys, slight hemorrhage in the thymus, slight congestion in the lungs, and breast carcinoma; the two euthanized animals from the 160 mg/kg group (5101, 5012) were observed with a mild increase in the amount of glycogen in the liver. There were observations of changes related to the administration of the test substance in the liver of animals subject to the scheduled sacrifice. This was a slight increase in the amount of glycogen accumulated in the hepatocytes for the 40 and 160 mg/kg group, which occurred in two animals from the 40 mg/kg group (4103, 4111) and seven animals from the 160 mg/kg group (5103, 5104, 5105, 5106, 5108, 5109, 5112). In addition, the HE-stained specimen of the liver cytoplasm from the 40 and 160 mg/kg group was found to become enlarged and lucent, leading to clearer appearance. In order to investigate the details future, PAS reaction and PAS reaction following saliva digestion were performed for two representative samples in the 160 mg/kg group (5101, 5108), which resulted in positive PAS reaction and negative PAS reaction after saliva digestion. Furthermore, increased extramedullary hematopoiesis was observed in the spleens of four animals from the control group (1101, 1104, 1105, 1112) and three animals from the 160 mg/kg group (5103, 5108, 5109).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
NOTE: Reproductive and developmental toxicity results are described under IUCLID Section 7.8.1.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
40 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
160 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
ovary
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
presumably yes
Conclusions:
The NOEL of the test item for general toxicology was thought to be 10 mg/kg bw/day for both males and females under the conditions of this study.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test conducted according to the OECD Test Guideline No. 422 and in compliance with GLP, the test item diluted in olive oil was administered to Crj:CD(SD) rat (12/sex/dose group) by gavage at dose levels of 0, 2.5, 10, 40 and 160 mg/kg bw/day. 


 


In male animals (P), the impact of test substance administration was not observed in the general condition, body weights, food consumption, hematology parameters, blood biochemistry parameters, necropsy findings, and organ weights. With regard to hematology and blood biochemistry, high mean corpuscular volume, low mean corpuscular hemoglobin concentration, prolonged prothrombin time, low chlorine, high A/G ratio and albumin ratio, and low γ-globulin ratio were observed in the 160 mg/kg bw/day group, with significant difference compared with the control group. However, a number of these parameters (mean corpuscular volume, mean corpuscular hemoglobin concentration, and chlorine) were within the normal range for Crj:CD rats, and only involved single parameters without changes in blood coagulation systems, increase in total protein, or other items suggesting the concentration of blood. In addition, the test substance was reported to cause hypocalcemia in rats and mice; however, the decrease in calcium as a blood coagulation factor was not observed in this study. With consideration for these findings, these changes were not thought to be caused by the administration of the test substance. Changes thought to be caused by the administration of the test substance were observed in the liver. The extent and frequency of the centrilobular fatty changes in the liver tended to decrease in the test substance groups at 40 mg/kg bw/day and higher. However, the mechanism of this change is unknown.


 


In female animals, no changes were observed in the general conditions during the pre-mating period and the mating period for any animals in the control group and the test substance groups. For changes in the general condition during the pregnancy period, deaths occurred in one maternal animal in the 40 mg/kg bw/day group during delivery on Day 22 of pregnancy and one maternal animal in the 160 mg/kg bw/day on Day 22 of pregnancy. No common findings considered to be directly related to death were observed in either animal as a result of histopathology performed on dead animals. No changes were observed in the general condition during the nursing period for any animals in the control group and the test substance groups. Also, the impact of test substance administration was not observed in the female animals with regard to body weights, food consumption, and necropsy findings. For organ weights, increase in liver weight and decrease in ovarian weight was observed for the 160 mg/kg bw/day group; however, no significant difference was observed between the test substance groups at 40 mg/kg bw/day and lower and the control group. Histopathological findings that are thought to be caused by the test substance administration, like males, were observed in the liver; however, the changes observed were different to those in the male animals. Slight increase in the amount of glycogen accumulated in the hepatocytes was observed for a small number of animals in the 40 mg/kg bw/day group and a large number of animals in the 160 mg/kg bw/day group. The HE-stained specimen of the liver cytoplasm from the 40 and 160 mg/kg bw/day group was found to become enlarged and lucent, leading to clearer appearance. In response to this, PAS reaction and PAS reaction after saliva digestion were performed, and the results suggested that the changes observed in the 40 and 160 mg/kg bw/day group reflected the distribution of glycogen in the cells.


 


As explained above, the impact of test substance administration was not observed in the male and female animals with regard to the general condition, body weights, food consumption, and necropsy findings. However, increase in liver weight and decrease in ovarian weight were observed for female animals in the 160 mg/kg bw/day group, and histopathological changes in the liver were observed in the 40 mg/kg bw/day and higher groups, although different findings were observed between males and females.


With consideration for the above, the NOEL of the test item for general toxicology was thought to be 10 mg/kg bw/day for both males and females under the conditions of this study.


 


This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable study is available. This study was conducted according to the OECD TG No. 422 (1996 version) under GLP compliance. In this study some deviations were noted compared to the current OECD TG No. 422 requirements which do not allow a complete assessment of the study results. However, the registered substance being a monomer imported only as a polymer in Europe there is no relevant human exposure. Therefore, further animal testing is not justified.
System:
hepatobiliary
Organ:
liver
ovary

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A key study was identified. In this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test conducted according to the OECD Test Guideline No. 422 and in compliance with GLP, the test item diluted in olive oil was administered to Crj:CD(SD) rat (12/sex/dose group) by gavage at dose levels of 0, 2.5, 10, 40 and 160 mg/kg bw/day. 


 


In male animals (P), the impact of test substance administration was not observed in the general condition, body weights, food consumption, hematology parameters, blood biochemistry parameters, necropsy findings, and organ weights. With regard to hematology and blood biochemistry, high mean corpuscular volume, low mean corpuscular hemoglobin concentration, prolonged prothrombin time, low chlorine, high A/G ratio and albumin ratio, and low γ-globulin ratio were observed in the 160 mg/kg bw/day group, with significant difference compared with the control group. However, a number of these parameters (mean corpuscular volume, mean corpuscular hemoglobin concentration, and chlorine) were within the normal range for Crj:CD rats, and only involved single parameters without changes in blood coagulation systems, increase in total protein, or other items suggesting the concentration of blood. In addition, the test substance was reported to cause hypocalcemia in rats and mice; however, the decrease in calcium as a blood coagulation factor was not observed in this study. With consideration for these findings, these changes were not thought to be caused by the administration of the test substance. Changes thought to be caused by the administration of the test substance were observed in the liver. The extent and frequency of the centrilobular fatty changes in the liver tended to decrease in the test substance groups at 40 mg/kg bw/day and higher. However, the mechanism of this change is unknown.


 


In female animals, no changes were observed in the general conditions during the pre-mating period and the mating period for any animals in the control group and the test substance groups. For changes in the general condition during the pregnancy period, deaths occurred in one maternal animal in the 40 mg/kg bw/day group during delivery on Day 22 of pregnancy and one maternal animal in the 160 mg/kg bw/day on Day 22 of pregnancy. No common findings considered to be directly related to death were observed in either animal as a result of histopathology performed on dead animals. No changes were observed in the general condition during the nursing period for any animals in the control group and the test substance groups. Also, the impact of test substance administration was not observed in the female animals with regard to body weights, food consumption, and necropsy findings. For organ weights, increase in liver weight and decrease in ovarian weight was observed for the 160 mg/kg bw/day group; however, no significant difference was observed between the test substance groups at 40 mg/kg bw/day and lower and the control group. Histopathological findings that are thought to be caused by the test substance administration, like males, were observed in the liver; however, the changes observed were different to those in the male animals. Slight increase in the amount of glycogen accumulated in the hepatocytes was observed for a small number of animals in the 40 mg/kg bw/day group and a large number of animals in the 160 mg/kg bw/day group. The HE-stained specimen of the liver cytoplasm from the 40 and 160 mg/kg bw/day group was found to become enlarged and lucent, leading to clearer appearance. In response to this, PAS reaction and PAS reaction after saliva digestion were performed, and the results suggested that the changes observed in the 40 and 160 mg/kg bw/day group reflected the distribution of glycogen in the cells.


 


As explained above, the impact of test substance administration was not observed in the male and female animals with regard to the general condition, body weights, food consumption, and necropsy findings. However, increase in liver weight and decrease in ovarian weight were observed for female animals in the 160 mg/kg bw/day group, and histopathological changes in the liver were observed in the 40 mg/kg bw/day and higher groups, although different findings were observed between males and females.


With consideration for the above, the NOEL of the test item for general toxicology was thought to be 10 mg/kg bw/day for both males and females under the conditions of this study.

Justification for classification or non-classification

Harmonised classification:


The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 


 


Self-classification: 


Based on the available information, no additional self-classification is proposed as there is no reliable evidence from the available study associating repeated exposure with a consistent and identifiable toxic effect.