Registration Dossier

Administrative data

Description of key information

Key, 28 -day Repeated dose toxicity, rat, oral (gavage), OECD 407, GLP: NOEL 20 mg/kg bw/day

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
Study period:
12 JAN 2001 - 30 MAY 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
other: MITI 1014
Version / remarks:
December 5, 1986
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan, Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 5 weeks
- Weight at study initiation: 134.1-153.8 g and 114.7-133.0 g for males and females
- Fasting period before study:
- Housing: hanging stainless steel cages with wire-mesh floor (one rat per cage, 165 Wx300 Dx150 H mm, TOKIWA KAGAKU KIKAI CO., LTD.)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 55±10%
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): light-dark cycle of 12-12 hours (light on: 7:00 and light off: 19:00)
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was melted in the original container by warming up in a hot water bath, and weighed accurately and dissolved with warm olive oil (Lot No. 013OOH, Fujimi Pharmaceutical Co., Ltd.) to make concentrations of 0.05, 0.2, 0.8 and 3.2 % (w/v).

VEHICLE
- Justification for use and choice of vehicle (if other than water): olive oil
- Concentration in vehicle: 0.05, 0.2, 0.8 and 3.2 w/v%
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Lot No. 013OOH
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC Analytical Conditions
Instrument (HP6890)
Date analyzer: HP GC-Chemstation (Hewlett packard), Gas chromatograph: HP6890 Series (Hewlett packard), Controller:G1512A (Hewlett packard), Injector:18593B (Hewlett packard)
Duration of treatment / exposure:
Treatment by oral gavage was carried out daily in the morning for 28 days, followed by observation for additional 14 days recovery period. A Nelaton Catheter (TERUMO CORPORATION) and a syringe (TERUMO CORPORATION) were used for dose administration.
Frequency of treatment:
daily for 28 days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
320 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight/day: 12 male, 12 female
Group 2: 5 mg/kg body weight/day : 6 male, 6 female
Group 3: 20 mg/kg body weight/day : 6 male, 6 female
Group 4: 80 mg/kg body weight/day : 6 male, 6 female
Group 5: 320 mg/kg body weight/day: 12 male, 12 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A 14-day preliminary repeated-dose oral toxicity study was carried out at three doses of 50,250 or 1,000 mg/kg/day. One female in the 1,000 mg/kg group died. Abnormalities were noted in blood chemical examinations in the 50, 250 and 1,000 mg/kg groups, in body weights, organ weights and histopathological examinations in the 250 and 1,000 mg/kg groups, and in clinical signs, hematological examination and necropsy in the 1,000 mg/kg group. Therefore, high dose level at 320 mg/kg/day and three lower doses at 80, 20 and 5 mg/kg/day were set for the main study. Recovery groups were prepared for the vehicle control and 320 mg/kg groups.
- Rationale for animal assignment (if not random): healthy animals with favorable weight gains were allocated to groups to ensure homogeneity of mean body weights using body weight-stratified randomization for the study.
- Fasting period before blood sampling for clinical biochemistry: overnight


Positive control:
no
Observations and examinations performed and frequency:
MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The day of the initiation of treatment was defined as day 1, and the preceding day as day -1. The week of the initiation of treatment was defined as week 1. Also, the next day of the final dosing was defined as recovery day 1, and the week of the initiation of recovery as recovery week 1.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed at least once daily for clinical signs

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed as follows:
Before Dosing: day -2 (at grouping)
During Dosing Period: day 1,3,8, 12, 17, 21, 26 and 28
During Recovery Period: day 1,5, 10 and 14
In addition, immediately before necropsy, body weights were measured for calculation of relative organ weights.

FOOD CONSUMPTION :
Food intakes were recorded as follows: Before Dosing: Once During Dosing period: day 3, 8, 15, 22 and 28 During Recovery Period: day 4, 8 and 14

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All survivors were fasted overnight (16-20 hours) at the termination of dosing and recovery periods, and blood samples were collected from abdominal aorta under ether anesthesia. Sodium citrate of 3.2% was used as an anticoagulant for examinations of prothrombin time and activated partial thromboplastin time, and EDTA-2K for other items.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all survicors
- Parameters:
1) Red Blood Cell Count (RBC)
2) White Blood Cell Count (WBC)
3) Hemoglobin Cone. (Hb)
4) Hematocrit Value (Ht)
5) Mean Corpuscular Volume (MCV)
6) Mean Corpuscular Hemoglobin (MCH)
7) Mean Corpuscular Hemoglobin Cone. (MCHC)
8) Platelet Count (Platelet)
9) Reticulocyte Count (Rcticulo)
10) Prothrombin Time (PT)
11) Activated Partial Thromboplastin Time (APTT)
12) Differentiation of Leukocytes
Stab Neutrophils (N-Band) Segmented Neutrophils (N-Seg) Eosinophils (Eosino) Basophils (Baso) Lymphocytes (Lymph) Monocytes (Mono)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Sera were separated from the blood samples collected at the same time of the hematological examinations and examined for following items. LDH and CPK were examined with the heparin treated samples
- Animals fasted: Yes
Parameters
1) GOT
2) GPT
3) Alkaline Phosphatase (ALP)
4) LDH
5) CPK
6) cholinesterase (ChE)
7) gamma-GTP
8) Total Cholesterol (T-Cho)
9) Triglyceride (TG)
10) Glucose
11) Total Protein (T-Protein)
12) Albumin
13) A/G Ratio
14) Blood Urea Nitrogen (BUN)
15) Creatinine
16) Total Bilirubin (T-Bil)
17) Calcium (Ca)
18) Inorganic Phosphorus (IP)
19) Sodium (Na)
20) Potassium (K)
21) Chloride (CI)

URINALYSIS: Yes
- Time schedule for collection of urine: Individual urine samples were collected in metabolic cages (200 W X 200 D X 380 Hmm) for 16 hr, at the periods, and examined for volume, color, and additional items of pH, protein, ketone bodies, bilirubin, occult blood, glucose and urobilinogen using a test paper (N-Multistix®, Bayer-Medical). Urinary sediment was examined in the vehicle control 80 and 320 mg/kg males and females, and 20 mg/kg females at end of the dosing period, and in the vehicle control and 320 mg/kg males and females at the end of the recovery period. termination of the dosing (day 28) and recovery (day 14)
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:
NECROPSY : YES
Animals were subjected to a detailed gross necropsy.

GROSS PATHOLOGY: YES
Organ Weights
Following organs were measured in wet weight:
Lungs, Liver, Heart, Kidneys, Testes, Ovaries, Brain, Spleen, Adrenals.

HISTOPATHOLOGY: Yes (see table)
1) The following organs and tissues were taken
Category Organs and Tissues
Respiratory system Trachea, Lungs
Digestive system Esophagus, Stomach, Intestine (Duodenum to Rectum), Pancreas, Liver
Cardiovascular system Heart
Urinary system Kidneys, Urinary bladder
Reproductive system Testes, Epididymides, Prostate, Seminal vesicle (with Coagulating gland), Ovaries, Uterus,Vagina
Nervous system Brain (Cerebrum, Cerebellum)
Hematopoietic and Lymphatic Bone marrow (Femur), Mesenteric lymph systems nodes, Spleen, Thymus, Mediastinal lymph nodes
Endocrine system Pituitary gland, Thyroids (with Parathyroids), Adrenals
Special sense organ Eye ball
The organs and tissues were preserved in 10% neutral buffered formalin. Testes and epididymides were fixed in Bouin's solution

2) Light microscopic examinations were performed for the following organs and tissues after embedding in paraffin, sectioning and hematoxylin and eosin staining. The histological preparation was conducted by Bio Pathology Institute, Ltd. according to the assignment protocol.
Group Organs and Tissues
Vehicle control group Lungs, Stomach, Tntestine (Duodenum, Jejunum, Ileum, group Cecum, Colon, Rectum), Liver, Heart, Kidneys, Urinary bladder, Ovaries, Uterus, Vagina, Mediastinal lymphnodes, Spleen, Adrenals
Vehicle control group (recovery) Lungs, Liver, Heart, Kidneys (females only), Mediastinal lymph nodes, Spleen (females only)
80 mg/kg group Lungs, Liver, Heart, Kidneys (females only),Mediastinal lymph nodes
320 mg/kg group Lungs, Stomach, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Liver, Heart, Kidneys, Urinary bladder, Ovaries, Uterus, Vagina, Mediastinal lymph nodes, Spleen, Adrenals
320 mg/kg group Lungs, Liver, Heart, Kidneys (females only), (recovery) Mediastinal lymph nodes, Spleen (females only)

(3) The following gross legions were examined histopathologically.
Group (Animal No.) Organs and tissues
5 mg/kg group (no. 15) Glandular stomach
20 mg/kg group (no.22) Glandular stomach
320 mg/kg group (recovery) (no.80) Glandular stomach

(4) The following organs and tissues were stained using other methods.
Group (Animal No.) Organs and tissues Method
80 mg/kg group (no. 28) Liver Oil red O
Other examinations:
FACTORS THAT MIGHT HAVE AFFECTED THE STUDY
1) Unforeseeable circumstances that might have affected the reliability of the study: In one female (no. 61) in the 20 mg/kg group, abnormal prothrombin time (PT) and activated partial thromboplastin time (APTT) were observed in the hematological examinations at the end of the dosing period. An accelerated coagulation was suggested in this case because of fibrin clot had been formed in the specimen. Given that this change was observed only in one animal in the 20 mg/kg group (the middle dose (1)) with no similar values observed in other animals, this change is likely to be an accidental change rather than a change related to the test article. Accordingly, the PT and APTT values of this animal were excluded from the statistical analysis. The above-mentioned unforeseeable circumstances that might have affected the reliability of the study were considered to have no effects on the results of the study.
2) Deviations from the protocol
With respect to the replacement of housing equipment for the housing environment, the protocol stipulated that racks would be replaced at a frequency of every 4 weeks; the rack replacement in week 4 was delayed by 2 days (i.e., once in 30 days). In the present final report, the frequency of the rack replacement is described as once a month.
3) There were no environmental factors that might have affected the reliability of the study results.
Statistics:
Data regarding body weights, food intakes, hematological examinations, blood chemical examinations, urine volume and organ weights were analyzed using the Bartlett's test for homogeneity of variance. If the variances were homogeneous at a significance level of 5%, one way analysis of variance was performed. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment groups was analyzed by the Dunnett's test.
If the variances were not homogeneous in the Kruskal-Wallis's test was used. If there was a significant difference in this analysis, the difference between the vehicle control group and each of the treatment group was analyzed by the nonparametric Dunnett's test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During Dosing Period
Male: Salivation was observed in the vehicle control group (7/12), 5 mg/kg group (6/6), 20 mg/kg group (4/6), 80 mg/kg group (4/6), and 320 mg/kg group (12/12).
Clinical aspects of salivation
Vehicle control group: Sporadically from day 8 to day 28, immediately after dosing.
5 mg/kg group: Sporadically or continuously from day 6 to day 28, immediately after dosing.
20 mg/kg group: Sporadically from day 6 to day 28, immediately after dosing.
80 mg/kg group: Sporadically or continuously from day 6 to day 28, immediately after dosing.
320 mg/kg group: Sporadically or continuously from day 6 to day 28, immediately after dosing.
Female: Salivation was observed in the vehicle control group (4/12), 5 mg/kg group (6/6), 20 mg/kg group (4/6), 80 mg/kg group (5/6), and 320 mg/kg group (10/12). Staining of the hair (1/12) were observed in the 320 mg/kg group in week 1.
Clinical aspects of salivation
Vehicle control group: Sporadically from day 6 to day 28, immediately after dosing.5
mg/kg group: Sporadically from day 6 to day 21, immediately after dosing.
20 mg/kg group: Sporadically from day 6 to day 28, immediately after dosing.
80 mg/kg group: Sporadically from day 6 to day 28, immediately after dosing.
320 mg/kg group: Sporadically or continuously from day 6 to day 28, immediately after dosing.
During Recovery Period: No abnormalities were noted in all groups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During Dosing Period: No abnormalities were noted in all groups.
During Recovery Period: No abnormalities were noted in all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During Dosing Period: No abnormalities were noted in all groups.
During Recovery Period: No abnormalities were noted in all groups.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Increased WBC was noted in the 80 mg/kg group. Increased WBC and platelet count were noted in the 320 mg/kg group.
Female: Increased WBC and a tendency toward increased platelet count were noted in the 320 mg/kg group. Decreased hemoglobin concentration, hematocrit value and APTT were noted in the 320 mg/kg group.
At Termination of Recovery Period
Male: No abnormalities were noted in all groups.
Female: Increased reticulocyte count was noted in the 320 mg/kg group. Decreased RBC, hemoglobin concentration, and hematocrit value were noted in the 320 mg/kg group. Increased segmented neutrophils ratio and decreased lymphocytes ratio were noted in the 320 mg/kg group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Increased creatinine level and tendencies toward increased LDH activity and BUN level were noted in the 320 mg/kg group. Decreased triglyceride level was noted in the 320 mg/kg.
Female: Increased GOT and LDH activities, increased BUN, creatinine and inorganic phosphorus levels, and decreased chloride level were noted in the 320 mg/ kg group.
At Termination of Recovery Period
Male: Increased GPT activity and inorganic phosphorus level, and decreased albumin level were noted in the 320 mg/kg group.
Female: Increased BUN and creatinine levels, and decreased chloride level were noted in the 320 mg/kg group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Increased white blood cells, epithelial cells and casts in the urinary sediment were noted in the 320 mg/kg group.
Female: A tendency toward positive reaction for occult blood was noted in the 320 mg/kg group. Increased epithelial cells in the urinary sediment were noted in the 80 mg/kg group. Increased white blood cells, epithelial cells and casts in the urinary sediment were noted in the 320 mg/kg group.
At Termination of Recovery Period
Male: No abnormalities were noted in all groups.
Female: Increased white blood cells and casts in the urinary sediment were noted in the 320 mg/kg group.
Behaviour (functional findings):
effects observed, treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Increased relative liver weight was noted in the 80 mg/kg group. Increased absolute and relative lung weights and relative liver weight were noted in the 320 mg/kg group.
Female: Increased relative liver weight was noted in the 80 mg/kg group. Increased absolute and relative lung, liver and kidney weights and relative heart weight were noted in the 320 mg/kg group. Increased relative brain weight and decreased absolute ovary and adrenal weights were noted in the 320 mg/kg group.
At Termination of Recovery Period
Male: Increased absolute and relative lung weights were noted in the 320 mg/kg group.
Female: Increased absolute and relative lung weights and increased relative liver, heart, kidney and spleen weights were noted in the 320 mg/kg group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Enlargement of the liver (1/6) was observed in the 80 mg/kg group. Pale change of the lungs (316), enlargement of the liver (5/6), and enlargement of the mediastinal lymph nodes (3/6) were observed in the 320 mg/kg group. Blackish region of the mucosa in the glandular stomach was observed in the vehicle control group (2/6), 5 mg/kg group (1/6), and 20 mg/kg group (1/6).Female: Pale change of the lungs (1/6), enlargement of the liver (6/6), enlargement (1/6) and pale change (3/6) of the kidneys, and enlargement of the mediastinal lymph nodes (5/6) were observed in the 320 mg/kg group.
At Termination of Recovery Period
Male: No abnormalities were noted in all groups.
Female: Blackish region of the mucosa in the glandular stomach (1/6) was observed in the 320 mg/kg group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Hypertrophy of the alveolar lining cells (+, 3/6 ; ++, 3/6) in the lungs, centrilobular hypertrophy (+, 1/6) and increased mitoses (+, 1/6) of the hepatocytes, focal myocarditis (+, 1/6), germinal center development (+, 1/6) and hypertrophy of the paracortex (+, 4/6) in the mediastinal lymph nodes were observed in the 320 mg/kg group. Necrosis of the fundic mucosa (+, 2/6) in the glandular stomach, focal myocarditis (+, 1/6), solitary cyst in medulla (+, 1/6) in the kidneys were observed in the vehicle control group. Necrosis of the fundic mucosa in the glandular stomach was observed in the 5 mg/kg group (+, 1/1) and 20 mg/kg group (+, 1/1). Diffuse lipid droplets (++, 1/6) and focal necrosis (+, 1/6) of the hepatocytes were observed in the 80 mg/kg group.
Female: Foamy cells, focal proliferation of the atypical pneumocytes, hypertrophy of the alveolar lining cells (+, 5/6) in the lung, centrilobular hypertrophy (±, 2/6 ; +, 3/6) of the hepatocytes, focal myocarditis (+, 3/6), atypical basophilic tubules (++, 6/6) and mineralization of the cortico-medullary junction (±, 1/6 ; +3/6) in the kidney, and hypertrophy of the paracortex (+, 4/6 ; ++, 1/6) in the mediastinal lymph node were observed in the 320 mg/kg group. Mineralization in the cortico-medullary junction (±, 1/6) and solitary cyst in medulla (+, 1/6) in the kidney were observed in the vehicle control group. Microgranuloma (+, 1/6) in the liver was observed in the 80 mg/kg group.
At Termination of Recovery Period
Male: Foamy cells (+, 5/6) and hypertrophy of the alveolar lining cells (+, 2/6) in the lung, focal myocarditis (±, 1/6), and hypertrophy of the paracortex (+, 1/6) in the mediastinal lymph node were observed in the 320 mg/kg group.
Female: Foamy cells (+, 6/6), and hypertrophy of the alveolar lining cells (+, 2/6) in the lung, atypical basophilic tubules (+, 3/6) and mineralization of the cortico-medullary junction (±, 1/6; +1/6) in the kidney, hypertrophy of the paracortex (+, 1/6) in the mediastinal lymph node, and increased extramedullary hematopoiesis (+, 2/6) in the spleen were observed in the 320 mg/kg group. Mineralization in the cortico-medullary junction (±, 2/6 ; +, 1/6) in the kidney was observed in the vehicle control group. Necrosis of the fluidic mucosa (+, 1/1) in the glandular stomach, microgranuloma (+, 1/6) in the liver and solitary cyst (+, 1/6) in medulla in the kidney were observed in the 320 mg/kg group.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
urinalysis
organ weights and organ / body weight ratios
gross pathology
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, the no-observed effect level (NOEL) of the test material in rats under the present experimental condition was estimated to be 20 mg/kg/day, because increased relative liver weight in males and females, increased WBC and enlargement of the liver in males, and increased epithelial cells in the urinary sediment in females were observed in the animals treated at 80 and 320 mg/kg/day.
Executive summary:

The oral sub-acute (28 days) repeated dose toxicity of the test item in rats has been examined according to OECD 407 and under GLP conditions.

Purpose

The purpose of this study is to define the type, severity and reversibility of toxicological signs and to determine the no-observed-effect-level (NOEL) of the test substance by observing the functional and morphological changes in animals receiving repeated doses orally for 28 days. This study should provide a rational basis for toxicological risk assessment in man.

Study Design

This study was conducted in accordance with "28-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances (Notification No. 700 of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) & No. 1014 (1986) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986)", and with "407, Repeated Dose Oral Toxicity-Rodent: 28-day or 14-day Study" prescribed in "OECD Guidelines for Testing of Chemicals (May 12, 1981)".

The oral 28-day repeated-dose toxicity study followed by a 14-day recovery period was performed in groups of 6 male and 6 female Crj:CD (SD) IGS rats at 5 weeks of age. Four dose levels were selected with the highest dose level set at 320 mg/kg, in addition to 80, 20, and 5 mg/kg. Recovery groups were set up with the animals treated at 320 mg/kg and the vehicle control.

Results

In the present study, neither deaths nor toxicologically significant changes in the clinical signs, body weights, and food intakes were observed during the dosing period.

The hematological examination findings observed at the end of the dosing period were as follows: increased white blood cell count (WBC) and platelet count in the 320 mg/kg males and females, decreased hemoglobin concentration (Hb), hematocrit value (Ht) and activated partial thromboplastin time (APTT) in the 320 mg/kg females; and increased WBC in the 80 mg/kg males. The blood chemical examination findings observed at the end of the dosing period were as follows: increased LDH activity, and blood urea nitrogen (BUN) and creatinine levels in 320 mg/kg males and females, decreased triglyceride (TG) level in the 320 mg/kg males, and increased GOT activity and inorganic phosphorus (IP) level and decreased chloride (Cl) level in the 320 mg/kg females. The urinalysis findings observed at the end of the dosing period were as follows: increased white blood cells, epithelial cells, and casts in the urinary sediment in the 320 mg/kg males and females; a tendency toward positive reaction for occult blood in the 320 mg/kg females; and increased epithelial cells in the urinary sediment in the 80 mg/kg females.

The findings of the organ weights observed at the end of the dosing period were as follows: increased relative liver weights in the 80 and 320 mg/kg males and females; increased absolute and relative lung weights in the 320 mg/kg males and females, and increased absolute and relative kidney weights, increased absolute liver weight, and increased relative heart weight in the 320 mg/kg females. Macroscopic examination findings observed at the end of the dosing period were as follows: pale change of the lungs, enlargement of the liver, and enlargement of the mediastinal lymph nodes in the 320 mg/kg males and females; enlargement and pale change of the kidneys in the 320 mg/kg females; and enlargement of the liver in the 80 mg/kg males. The histopathological examination findings observed at the end of the dosing period were as follows: hypertrophy of alveolar lining cells in the lungs, centrilobular hypertrophy of hepatocytes, focal myocarditis, and hypertrophy of the paracortex in the mediastinal lymph nodes in the 320 mg/kg males and females; increased mitoses of hepatocytes and germinal center development in the mediastinal lymph nodes in the 320 mg/kg males, and foamy cells and focal proliferation of atypical pneumocytes in the lungs, atypical basophilic tubules, and mineralization of the cortico-medullary junction in the kidney in the 320 mg/kg females.

The findings observed at the end of the recovery period in the recovery study were as follows: increased absolute and relative lung weights and hypertrophy of alveolar lining cells in the lungs, hypertrophy of paracortex in the mediastinal lymph nodes in the 320 mg/kg males and females, increased BUN and creatinine levels, decreased hemoglobin concentration, hematocrit value, and chloride level, increased white blood cells and casts in the urinary sediment, increased relative liver, heart and kidney weights, foamy cells in the lungs, atypical basophilic tubules and mineralization of the cortico¬medullary junction in the kidneys in the 320 mg/kg females. In addition, foamy cells in the lungs in males, and decreased red blood cell count (RBC), increased reticulocytes count, increased relative spleen weight, and increased extramedullary hematopoiesis in the spleen in females were observed in the 320 mg/kg groups as new changes. Most changes in the organs and tissues that showed histopathological changes at the end of the dosing period, however, were considered to be reversible.

Conclusions

Based on the results of this study, the no-observed effect level (NOEL) of the test material in rats under the present experimental condition was estimated to be 20 mg/kg/day, because increased relative liver weight in males and females, increased WBC and enlargement of the liver in males, and increased epithelial cells in the urinary sediment in females were observed in the animals treated at 80 and 320 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
high quality due to guideline study and GLP compliance.
System:
hepatobiliary
Organ:
heart
kidney
liver
lungs

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

Repeated-dose toxicity_oral_subacute

The oral subacute (28 days) repeated dose toxicity of the test item in rats has been examined according to OECD 407 and under GLP conditions.

Purpose

The purpose of this study is to define the type, severity and reversibility of toxicological signs and to determine the no-observed-effect-level (NOEL) of the test substance by observing the functional and morphological changes in animals receiving repeated doses orally for 28 days. This study should provide a rational basis for toxicological risk assessment in man.

Study Design

This study was conducted in accordance with "28-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances (Notification No. 700 of the Planning and Coordination Bureau, Environment Agency (EA), No. 1039 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare (MHW) & No. 1014 (1986) of the Basic Industries Bureau, Ministry of International Trade and Industry (MITI), December 5, 1986)", and with "407, Repeated Dose Oral Toxicity-Rodent: 28-day or 14-day Study" prescribed in "OECD Guidelines for Testing of Chemicals (May 12, 1981)".

The oral 28-day repeated-dose toxicity study followed by a 14-day recovery period was performed in groups of 6 male and 6 female Crj:CD (SD) IGS rats at 5 weeks of age. Four dose levels were selected with the highest dose level set at 320 mg/kg, in addition to 80, 20, and 5 mg/kg. Recovery groups were set up with the animals treated at 320 mg/kg and the vehicle control.

Results

In the present study, neither deaths nor toxicologically significant changes in the clinical signs, body weights, and food intakes were observed during the dosing period.

The hematological examination findings observed at the end of the dosing period were as follows: increased white blood cell count (WBC) and platelet count in the 320 mg/kg males and females, decreased hemoglobin concentration (Hb), hematocrit value (Ht) and activated partial thromboplastin time (APTT) in the 320 mg/kg females; and increased WBC in the 80 mg/kg males. The blood chemical examination findings observed at the end of the dosing period were as follows: increased LDH activity, and blood urea nitrogen (BUN) and creatinine levels in 320 mg/kg males and females, decreased triglyceride (TG) level in the 320 mg/kg males, and increased GOT activity and inorganic phosphorus (IP) level and decreased chloride (Cl) level in the 320 mg/kg females. The urinalysis findings observed at the end of the dosing period were as follows: increased white blood cells, epithelial cells, and casts in the urinary sediment in the 320 mg/kg males and females; a tendency toward positive reaction for occult blood in the 320 mg/kg females; and increased epithelial cells in the urinary sediment in the 80 mg/kg females.

The findings of the organ weights observed at the end of the dosing period were as follows: increased relative liver weights in the 80 and 320 mg/kg males and females; increased absolute and relative lung weights in the 320 mg/kg males and females, and increased absolute and relative kidney weights, increased absolute liver weight, and increased relative heart weight in the 320 mg/kg females. Macroscopic examination findings observed at the end of the dosing period were as follows: pale change of the lungs, enlargement of the liver, and enlargement of the mediastinal lymph nodes in the 320 mg/kg males and females; enlargement and pale change of the kidneys in the 320 mg/kg females; and enlargement of the liver in the 80 mg/kg males. The histopathological examination findings observed at the end of the dosing period were as follows: hypertrophy of alveolar lining cells in the lungs, centrilobular hypertrophy of hepatocytes, focal myocarditis, and hypertrophy of the paracortex in the mediastinal lymph nodes in the 320 mg/kg males and females; increased mitoses of hepatocytes and germinal center development in the mediastinal lymph nodes in the 320 mg/kg males, and foamy cells and focal proliferation of atypical pneumocytes in the lungs, atypical basophilic tubules, and mineralization of the cortico-medullary junction in the kidney in the 320 mg/kg females.

The findings observed at the end of the recovery period in the recovery study were as follows: increased absolute and relative lung weights and hypertrophy of alveolar lining cells in the lungs, hypertrophy of paracortex in the mediastinal lymph nodes in the 320 mg/kg males and females, increased BUN and creatinine levels, decreased hemoglobin concentration, hematocrit value, and chloride level, increased white blood cells and casts in the urinary sediment, increased relative liver, heart and kidney weights, foamy cells in the lungs, atypical basophilic tubules and mineralization of the cortico¬medullary junction in the kidneys in the 320 mg/kg females. In addition, foamy cells in the lungs in males, and decreased red blood cell count (RBC), increased reticulocytes count, increased relative spleen weight, and increased extramedullary hematopoiesis in the spleen in females were observed in the 320 mg/kg groups as new changes. Most changes in the organs and tissues that showed histopathological changes at the end of the dosing period, however, were considered to be reversible.

Conclusions

Based on the results of this study, the no-observed effect level (NOEL) of the test material in rats under the present experimental condition was estimated to be 20 mg/kg/day, because increased relative liver weight in males and females, increased WBC and enlargement of the liver in males, and increased epithelial cells in the urinary sediment in females were observed in the animals treated at 80 and 320 mg/kg/day.

Justification for classification or non-classification

Based on the provided information the test item is not classified as STOT RE according to the Regulation (EC) No 1272/2008.


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