Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 16, 2000 - March 8, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
OECD Guidelines for Testing of Chemicals (May 12, 1981)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Batch: E004115

Test animals

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: yes
- Age at study initiation: 5 weeks
- Weight at study initiation: 124.0-143.5 g (males), 105.0-130.8 g (females)
- Fasting period before study: no
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 d

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 55±10%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard vehicle showing best solubility, homogenicity and stability
- Concentration in vehicle: 1.5 w/v% and subsequent dilutions
- Amount of vehicle (if gavage): 10 mL / kg
- Lot/batch no. (if required): 014OOY, Fujimi Pharmaceutical Co., Ltd.
- Purity: no data
- Rate of preparation: once weekly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC Analytical Conditions
(1) Instrument (HP6890)
Data analyzer: HP GC-Chemstation, Hewlett packard
Gas chromatograph: HP 6890 Series, Hewlett packard
Controller: G1512A, Hewlett packard
Injector: 18593B, Hewlett packard
(2) Condition
Column: HP-1 (F.T.0.25 µm) 0.32 mm i.d.  30 m
Oven temperature: 150ºC (0 min)→15ºC/min→280ºC (0 min)
Injection temperature: 250ºC
Detector: FID
Detector temperature: 250ºC
Injected amount: 1 µl
Injection method: Splitless
Carrier gas: Helium
Carrier gas flow rate: 1.0 ml/min
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily (7 d/week)
Doses / concentrationsopen allclose all
Dose / conc.:
6 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
0 mg/kg bw/d: 24 (12 main kill, 12 recovery)
6 mg/kg bw/d 12
30 mg/kg bw/d: 12
150 mg/kg bw/d: 24 (12 main kill, 12 recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: n.a.
- Post-exposure recovery period in satellite groups: n.a.
- Section schedule rationale (if not random): random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations:
All animals were weighed as follows:
Before Dosing: day -1 (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 AND COMPOUND INTAKE (if feeding study):
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



HAEMATOLOGY: Yes
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. Thin bone marrow smears were prepared from left femurs in the first 3 animals of each group at the terminal necropsy of the dosing and recovery periods, and examined in both sexes of the high dose and vehicle control groups at the terminal necropsy of the dosing period. 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.
Parameters Method
-----------------------------------------------------------------------------------------------------------
1) Red Blood Cell Count (RBC) (×10^4/µl) System for detecting change
in electrical resistance
2) White Blood Cell Count (WBC) (×10^2/µl) System for detecting change
in electrical resistance
3) Hemoglobin Conc. (Hb) (g/dl) Noncyanhemoglobin method
4) Hematocrit Value (Ht) (%)
5) Mean Corpuscular Volume (MCV) (fl) System for detecting change
in electrical resistance
6) Mean Corpuscular Hemoglobin (MCH) (pg)
7) Mean Corpuscular Hemoglobin Conc. (MCHC) (g/dl)
8) Platelet Count (Platelet) (×104/µl) System for detecting change
in electrical resistance
9) Reticulocyte Count (Reticulo) (‰) New methylene blue staining
10) Prothrombin Time (PT) (sec) Magnetic sensor system
11) Activated Partial Thromboplastin Time (APTT) (sec) Magnetic sensor system
12) Fibrinogen Conc. (Fbg) (mg/dl) Thrombin time method
13) Differentiation of Leukocytes (%) Wright staining
Stab Neutrophils (N-Band)
Segmented Neutrophils (N-Seg)
Eosinophils (Eosino)
Basophils (Baso)
Lymphocytes (Lymph)
Monocytes (Mono)
Myelogram (%) May-Grünwald-Giemsa staining
Myelobrasts (MyBl) (500 cells)
Promyelocytes (PMy)
Myelocytes, MetaMyelocytes (My, Mt)
Stab neutrophils, Segmented neutrophils (N-Band, N-Seg)
Eosinpphils (Eosino)
Basophils (Baso)
Lymphocytes (Lymph)
Plasmacytes (Plas)
Megakaryocytes (Mk)
Retoperitheliums (Ret)
Mastocytes (Mast)
Monocytes (Mono)
Proerythroblasts, Basoerythroblasts (PEb, BEb)
Polychromatic erythroblasts, neuerythroblasts (PoEb, NEb)
Myeloblast series/Erythroblasts series (M/E ratio)
-----------------------------------------------------------------------------------------------------------
1) - 8) Hematology Analyzer, CELL-DYN3500, Abbott Laboratories
9), 13) Automatic Blood Cell Analyzer, MICROX HEG-120A, OMRON Corporation
10) - 12) Coagulometer, KC-10A, AMELUNG
14) Microscope, OLYMPUS
-----------------------------------------------------------------------------------------------------------



CLINICAL CHEMISTRY: Yes
Sera were separated from the blood samples collected at the same time of the hematological examinations and examined for following items.
Parameters Method
1) GOT (IU/l) UV method (Method based on JSCC)
2) GPT (IU/l) UV method (Method based on JSCC)
3) Alkaline Phosphatase (ALP) (IU/l) p-Nitrophenyl phosphate method
4) Cholinesterase (ChE) (IU/l) Butyrylthiocholine iodide method
5) -GTP (IU/l) L--Glutamyl-p-nitroanilide method
6) Total Cholesterol (T-Cho) (mg/dl) COD·ADPS method
7) Triglyceride (TG) (mg/dl) GPO·ADPS glycerol blocking method
8) Glucose (mg/dl) Hexokinase·G-6-PDH method
9) Total Protein (T-Protein) (g/dl) Biuret method
10) Albumin (g/dl) Bromocresol green method
11) A/G Ratio
12) Blood Urea Nitrogen (BUN) (mg/dl) Urease·GlDH method
13) Creatinine (mg/dl) Creatininase·F-DAOS method
14) Total Bilirubin (T-Bil) (mg/dl) Azobilirubin method
15) Calcium (Ca) (mg/dl) OCPC method
16) Inorganic Phosphorus (IP) (mg/dl) Fiske-Subbarow method
17) Sodium (Na) (mEq/l) Crown-Ether membrane
electrode method
18) Potassium (K) (mEq/l) Crown-Ether membrane
electrode method
19) Chloride (Cl) (mEq/l) Coulometric titration method
-----------------------------------------------------------------------------------------------
1) - 3), 6)-8), 12), 13), 15), 16) 7170 Automatic Analyzer, Hitachi, Ltd.
4), 5), 9), 10), 14) 7150 Automatic Analyzer, Hitachi, Ltd.
17) - 19) PVA-III, A & T
-----------------------------------------------------------------------------------------------



URINALYSIS: Yes
Individual urine samples were collected in metabolic cages (200 W×200 D×380 H mm) for 16 hr, at the termination of the dosing (day 28) and recovery (day 14) 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 high dose and the vehicle control groups at the termination of the dosing period.


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:


Sacrifice and pathology:
Histological Examinations
- Necropsy
Animals were subjected to a detailed gross necropsy.
- Organ Weights
Following organs were measured in wet weight:
Liver, Heart, Kidneys, Testes, Epididymides, Prostate, Seminal vesicle, Ovaries, Uterus, Brain, Spleen, Thymus, Pituitary gland, Thyroids, Adrenals
- The following organs and tissues were taken.
-----------------------------------------------------------------------------------------------
Category Organs and Tissues
-----------------------------------------------------------------------------------------------
Respiratory system Lungs
Digestive system Stomach, Intestine (Duodenum to Rectum with Peyer’s patches), 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 and Cerebellum)
Hematopoietic and Lymphatic systems Bone marrow (Femur), Spleen, Thymus
Endocrine system Pituitary gland, Thyroids (with Parathyroids), Adrenals
Special sense organ Eye ball
Skin Mammary glands
Others Gross Lesions
-----------------------------------------------------------------------------------------------
The organs and tissues were preserved in 10% neutral buffered formalin. Testes and epididymides were fixed in Bouin’s solution.


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, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Liver, Heart, Kidneys, Testes, Epididymides, Prostate, Coagulating gland, Seminal vesicle, Ovaries, Uterus, Vagina, Spleen, Thymus, Pituitary gland, Thyroids, Adrenals, Mammary glands
-----------------------------------------------------------------------------------------------
Vehicle control
group (recovery) Lungs, Glandular stomach (females only), Liver, Kidneys (females only), Testes, Epididymides, Ovaries, Uterus, Vagina, Spleen, Thyroids (females only), Adrenals
-----------------------------------------------------------------------------------------------
6 mg/kg group Liver, Ovaries, Uterus, Vagina, Adrenals
-----------------------------------------------------------------------------------------------
30 mg/kg group Lungs, Glandular stomach (females only), Liver, Testes, Epididymides, Ovaries, Uterus, Vagina, Adrenals
-----------------------------------------------------------------------------------------------
150 mg/kg group Lungs, Stomach, Intestine (Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Liver, Heart, Kidneys, Testes, Epididymides, Prostate, Coagulating gland, Seminal vesicle, Ovaries, Uterus, Vagina, Spleen, Thymus, Pituitary gland, Thyroids, Adrenals, Mammary glands
-----------------------------------------------------------------------------------------------
150 mg/kg group (recovery) Lungs, Glandular stomach (females only), Liver, Kidneys (females only), Testes, Epididymides, Ovaries, Uterus, Vagina, Spleen, Thyroids (females only), Adrenals
-----------------------------------------------------------------------------------------------


The following gross regions were examined histopathologically.
-----------------------------------------------------------------------------------------------
Group (Animal No.) Organs and tissues
-----------------------------------------------------------------------------------------------
Vehicle control group (no. 1) Skin
-----------------------------------------------------------------------------------------------
Vehicle control group (no. 17) Adrenals
-----------------------------------------------------------------------------------------------
150 mg/kg group
(nos. 61, 63, 64, 65, 66) Skin
-----------------------------------------------------------------------------------------------
150 mg/kg group (recovery)
(nos. 67, 68, 71, 72) Skin
-----------------------------------------------------------------------------------------------

The following organs and tissues were stained using other methods.
-----------------------------------------------------------------------------------------------
Group (Animal No.) Organs and tissues Method
-----------------------------------------------------------------------------------------------
Vehicle control group
(nos. 6, 37) Adrenals Oil red O, Nile blue
-----------------------------------------------------------------------------------------------
150 mg/kg group
(no. 29, 61) Adrenals Oil red O, Nile blue
-----------------------------------------------------------------------------------------------
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During Dosing Period
Males:
Salivation (8/12), loss of the hair (1/12), scab formation (1/12), and exudate (1/12) on the neck were observed in the vehicle control group. Salivation was observed in the 6 mg/kg group (6/6), 30 mg/kg group (6/6), and 150 mg/kg group (12/12). Soft stool (2/12) was observed in the 150 mg/kg group.
Females:
Salivation was observed in the vehicle control group (3/12), 6 mg/kg group (3/6), 30 mg/kg group (5/6), and 150 mg/kg group (12/12). Soft stool (5/12), decreased stool volume (3/12), staining around the nose and mouth (2/12), emaciation (1/12), nasal bleeding (2/12), unkempt hair (3/12), decreased spontaneous locomotion (5/12), decreased respiratory rate (4/12), subnormal temperature (1/12), cyanosis (1/12), white turbid urine (1/12), lacrimation (4/12), staining around the anus (1/12), cage biting (1/12), cage licking (1/12), lid closure (2/12), irritability (1/12), tremor (1/12), staggering gait (1/12), and loss of hair (8/12) and moist hair (3/12) around the urogenital region were observed in the 150 mg/kg group. Cage biting, irritability and tremor were observed after dosing on day 18. Staggering gait was observed before dosing on day 19. Cage licking was observed in one animal (no. 67) immediately after dosing on day 19.

During Recovery Period
Male: No abnormalities were noted in all groups.
Female: Decreased stool volume (1/6), staining around the nose and mouth (1/6), emaciation (1/6), unkempt hair (2/6), decreased spontaneous locomotion (1/6), lacrimation (1/6), loss of hair around the urogenital region (4/6), staining in the lower abdomen (1/6), and mucous stool (1/6) were observed in the 150 mg/kg group.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During Dosing Period
Male: Lower body weights in the 150 mg/kg group were observed when compared to the vehicle control on day 12 - day 28.
Female: Lower body weights in the 150 mg/kg group were observed when compared to the vehicle control on day 8 - day 28.
During Recovery Period
Male: Lower body weights in the 150 mg/kg group were observed when compared to the vehicle control on day 1 - day 14.
Female: Lower body weights in the 150 mg/kg group were observed when compared to the vehicle control on day 1 - day 14.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During Dosing Period
Male: Decreased food intakes were noted in the 150 mg/kg group on day 15.
Female: Decreased food intakes were noted in the 150 mg/kg group on day 3 - day 28.
During Recovery Period
Male: Decreased food intakes were noted in the 150 mg/kg group on day 8 and day 14.
Female: Decreased food intakes were noted in the 150 mg/kg group on day 4 and day 8.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Increased RBC, hemoglobin concentration, and hematocrit value and decreased platelet count were noted in the 150 mg/kg group. Increased hemoglobin concentration and hematocrit value were noted in the 6 mg/kg group.
Female: Increased RBC, hemoglobin concentration, and hematocrit value and a tendency toward decreased platelet count were noted in the 150 mg/kg group.

At Termination of Recovery Period
Male: Increased prothrombin time was noted in the 150 mg/kg group.
Female: Decreased MCHC and increased reticulocyte count were noted in the 150 mg/kg group
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Decreased triglyceride levels were noted in the 6, 30 and 150 mg/kg groups. Increased GOT, GPT, alkaline phosphatase, and cholinesterase activities, total cholesterol, and total birilubine levels were noted in the 150 mg/kg group.
Female: Tendencies toward decreased triglyceride levels were noted in the 6 and 30 mg/kg groups. Increased GPT activities were noted in the 30 and 150 mg/kg groups. Increased alkaline phosphatase activity, total birilubine and potassium levels were noted in the 150 mg/kg group. Tendencies toward increased GOT and gamma-GTP activities, decreased cholinesterase activity, A/G ratio, total cholesterol, triglyceride, creatinine, and calcium levels were noted in the 150 mg/kg group. Decreased GOT activity was noted in the 6 mg/kg group.

At Termination of Recovery Period
Male: Increased gamma-GTP activity and decreased triglyceride level were noted in the 150 mg/kg group.
Female: Increased alkaline phosphatase and gamma-GTP activities and decreased triglyceride and calcium levels were noted in the 150 mg/kg group.

Urinalysis findings:
no effects observed
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):
At Termination of Dosing Period
Male: Increased relative liver weights were noted in the 30 and 150 mg/kg groups. Increased relative heart weight, decreased absolute epididymis weights, increased absolute and relative adrenals weights, and a tendency toward decreased absolute pituitary gland weight were noted in the 150 mg/kg group.
Female: Increased relative liver weights were noted in the 30 and 150 mg/kg groups. Increased absolute liver weight and increased absolute and relative adrenals weights were noted in the 150 mg/kg group. Increased absolute and relative pituitary gland weights were noted in the 6 mg/kg group. Increased relative brain weight was noted in the 150 mg/kg group.

At Termination of Recovery Period
Male: Decreased absolute epididymis and pituitary gland weights, and increased relative prostate, spleen and adrenals weights were noted in the 150 mg/kg group. Increased relative brain weight was noted in the 150 mg/kg group.
Female: Increased relative liver and adrenals weights were noted in the 150 mg/kg group. Increased relative kidneys, thyroid and brain weights were noted in the 150 mg/kg group.

Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At Termination of Dosing Period
Male: Loss of hair (1/6) was observed in the vehicle control group. Blackish region of the mucosa (1/6) in the glandular stomach was observed in the 6 mg/kg group.
Female: Enlargement of the liver (6/6), enlargement of the adrenals (6/6) and loss of hair (5/6) were observed in the 150 mg/kg group. Small ovaries (1/6) were observed in the vehicle control group.

At Termination of Recovery Period
Male: Brackish change of the spleen (1/6) was observed in the 150 mg/kg group.
Female: Blackish change of the spleen (1/6) and loss of hair (4/6) were observed in the 150 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: Centrilobular hypertrophy (+, 3/6) of the hepatocytes and coarse vacuolization (+, 2/6) and fine vacuolization (+, 4/6) of the zona fasciculate in the adrenal were observed in the 30 mg/kg group. Formy cells (+, 1/6) in the lung, centrilobular hypertrophy (+, 6/6) of the hepatocytes, deep retention of the spermatides (+, 1/6), inhibited spermiation (+, 1/6), multinucleated giant cell formation (+, 1/6) and vacuolization of Leydig cells (±, 2/6; +, 2/6) in the testis, decreased spermatozoa (+, 1/6) and germ cell debris (±, 2/6) in the lumen of the epididymis, and coarse vacuolization (+, 3/6; ++, 1/6) and fine vacuolization (+, 4/6; ++, 2/6) of the zona fasciculate in the adrenal were observed in the 150 mg/kg group. Round cell infiltration (+, 1/6) in the prostate and decreased hair follicles (+, 1/1) were observed in the vehicle control group. Necrosis of the fundic mucosa (+, 1/1) in the glandular stomach was observed in the 6 mg/kg group. Round cell infiltration (+, 1/6) in the prostate were observed in the 150 mg/kg group.
Female: Centrilobular hypertrophy of the hepatocytes (+, 3/6), fine vacuolization of the corpora (±, 1/6) and the interstitial gland (±, 3/6; +, 2/6) in the ovary, and fine vacuolization of the zona fasciculata (±, 3/6; ++, 3/6) in the adrenal were observed in the 30 mg/kg group. Formy cells (+, 1/6) in the lung, necrosis of the pyloric mucosa (+, 1/6) in the glandular stomach, centrilobular hypertrophy (++, 6/6) of the hepatocytes, fine vacuolization of the corpora lutea (+, 3/6) and the interstitial gland (+, 3/6; ++, 3/6) in the ovary, and fine vacuolization of the zona fasciculata (+++, 6/6) in the adrenal were observed in the 150 mg/kg group. Mineralization in the corticomedullary junction (+, 1/6) in the kidney, and absence of the oocytes (1/6) in the ovary were observed in the vehicle control group.

At Termination of Recovery Period
Male: Foamy cells (+, 1/6) in the lung, congestion (+, 1/6) in the spleen and coarse vacuolization (+, 1/6; ++, 1/6) and fine vacuolization (+, 2/6) of the zona fasciculate in the adrenal were observed in the 150 mg/kg group.
Female: Centrilobular hypertrophy (+, 3/6) of the hepatocytes, fine vacuolization of the corpora lutea (+, 1/6) in the ovary, congestion (+, 1/6) and increased extramedulalry hematopoiesis (+, 2/6) in the spleen, and fine vacuolization of the zona fasciculate (±, 2/6) in the adrenal was observed in the 150 mg/kg group. Mineralization in the corticomedullary junction were observed in the vehicle control group (+, 1/6) and 150 mg/kg group (+, 1/6).

In the adrenals in the 150 mg/kg group given special staining, increased positive (red) substance in the Oil red O staining or increased light red - red substance in the Nile Blue staining were observed in the adrenal cortex, suggestive that the fine vacuolization of the zona fasciculata observed in preparations stained with hematoxylin and eosin is caused by an accumulation of neutral lipids.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these results, the effects of the test material were observed as histopathological changes in the genital system, adrenals, liver, lungs, and stomach, suggesting effects on the production of steroid hormones in the adrenals and the genital system.  In the recovery study, although reversibility was observed in the liver, lungs, and stomach, no reversibility was observed in the adrenals or the genital system.  Accordingly, the NAOEL of the test material under the present experimental condition was suggested to be 6 mg/kg/day, based on clinical biochemistry alterations (TG levels) without any other adverse findings (histopathology, clinical signs).
Executive summary:

Due to the low severity of the effects, the test item is not considered to be classified for STOT RE according to the Regulation (EC) No 1272/2008.