Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan 12 - May 30, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted according to Good Laboratory Practice (GLP). 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)".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: MITI 1014
Principles of method if other than guideline:
None
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: CCP-V-1
Other name: RO-4562
Description: solid, off white, crystal mass with a yellowish tinge
Batch number: 200517
Purity: 99.98 area% (HPLC)
Stability of test item: Stable under storage conditions
Expiry date: 30. April 2007
Storage conditions: at room temperature (15-25°C), tightly closed, protected from light and moisture
Safety precautions: Routine hygienic procedures

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS
Crj: CD (SD) IGS rats (SPF) were obtained from Charles River Japan, Inc. (Hino Breeding Center; 735, Shimokomatsuki, Hino-cho, Gamo-gun, Shiga 529-1633, Japan). Animals were quarantined and acclimatized, and 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. At the onset of the treatment, the animals were 5 weeks old with body weight ranges of 134.1-153.8 g and 114.7-133.0 g for males and females, respectively. Animals were identified by ear-tagging.

HOUSING CONDITIONS
The barrier-system animal room was maintained at a stable temperature (23±2°C) and relative humidity (55±10%) with 10-15 air changes per hour and artificial light-dark cycle of 12-12 hours (light on: 7:00 and light off: 19:00). The rats were housed in hanging stainless steel cages with wire-mesh floor (one rat per cage, 165 Wx300 Dx150 H mm, TOKIWA KAGAKU KIKAI CO., LTD.). Trays were changed twice a week, cages once a week and rack once a month. The racks and cages were identified by individual cards. The animals had free access to an MF pelleted diet (Oriental Yeast, Co., Ltd.) and water (chlorinated) from the Hita City supply via automatic watering system with sipper tubes. The diet and housing materials were autoclaved at 121°C for 30 min prior to use. Analysis of contaminants in both the diet and drinking water confirmed that they would not affect the test system.






Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
Rationale for dosage selection:
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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC Analytical Conditions
(1) Instrument (HP6890)
Date analyzer: HP GC-Chemstation (Hewlett packard) Gas chromatograph: HP6890 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. x 30 m
Oven temperature: 150°C(0 min)-*20°C /min-X290°C (0 min)
Injection temperature: 300°C
Detector: FID
Detector temperature: 300°C
Injected amount: 1µl
Injection method: Splitless
Carrier gas: Helium
Carrie gas flow rate: 0.9 mllmin
Duration of treatment / exposure:
Treatment by oral gavege 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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 20, 80, 320 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Group 1: 0 mg/kg body weight/day: 12
Group 2: 5 mg/kg body weight/day : 6
Group 3: 20 mg/kg body weight/day : 6
Group 4: 80 mg/kg body weight/day : 6
Group 5: 320 mg/kg body weight/day: 12
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
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.

CLINICAL SIGNS
All animals were observed at least once daily for clinical signs.

BODY WEIGHT
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 INTAKE
Before Dosing: Once per day
During Dosing period: day 3, 8, 15, 22 and 28
During Recovery Period: day 4, 8 and 14

HEMATOLOGICAL INFORMATION
1) Red Blood Cell Count (RBC)
2) White Blood Cell Count (WBC)
3) Hemoglobin Conc. (Hb)
4) Hematocrit Value (Ht)
5) Mean Corpuscular Volume (MCV)
6) Mean Corpuscular Hemoglobin (MCH)
7) Mean Corpuscular Hemoglobin Conc. (MCHC)
8) Platelet Count (Platelet)
9) Reticulocyte Count (Reticulo)
10) Prothrombin Time (PT)
11) Activated Partial Thrornboplastin Time (APTT)
12) Differentiation of Leukocytes
(Stab Neutrophils (N-Band), Segmented Neutrophils (N-Seg), Eosinophils (Eosin), Basophils (Baso), Lymphocytes (Lymph), Monocytes (Mono))


CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined:
Parameters
1) GOT
2) GPT
3) Alkaline Phosphatase (ALP)
4) LDH
5) CPK
6) Cholinesterase (ChE)
7) y-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 (Cl)

Urinalysis
Individual urine samples were collected in metabolic cages (200 W X 200 D X 380 Hmm) 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 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.


Necropsy
Animals were subjected to a detailed gross necropsy.

Organ Weights
Following organs were measured in wet weight:
Lungs, Liver, Heart, Kidneys, Testes, Ovaries, Brain, Spleen, Adrenals
Sacrifice and pathology:
NECROPSY
Sacrifice:
after 4 weeks 28-July-2006 (groups 1-3, Allocation A)
after 8 days 07-July-2006 (group 4, Allocation A and B)
after 6 weeks 11-Aug-2006 (group 1, Allocation B)
after 6 weeks 01-Sept-2006 (group 3, Allocation B)
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination. From group 4 animals (allocation A and B), blood samples were taken by heart puncture at necropsy if possible. No blood for determination of coagulation parameters was collected from these animals.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated):
Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain (at least 3 levels)
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes w/optic nerve (fixed in Davidson's solution)
Harderian gland (fixed in Davidson's solution)
Heart
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland, exorbital
Liver
Lungs, filled w/formalin at necropsy
Lymph nodes - mesenteric, mandibular
Mammary gland area
Nasal cavity
Ovaries
Pancreas
Pituitary gland
Prostate gland (incl. coagulating gland)
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Urinary bladder, filled w/formalin at necropsy
Uterus
Vagina
Gross lesions
Additional tissues (such as ear tattoo) were retained in accordance with standard operating procedures but were not processed or examined further.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:

Brain Thymus Spleen Ovaries Heart Kidneys Testes Liver Adrenals Epididymides

The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) that were collected at terminal sacrifice from the animals of the control and group 3 were examined by the study pathologist.
Since test item-related morphologic changes were detected in organs of the high-dose (group 3) animals, those same organs (liver, adrenal glands, and testes) from the low-dose group (group 2) were examined to establish a no-effect level.
The same organs of group 4 animals which were terminated in extremis after 6 days of treatment on day 8 (July, 7th, 2006) were not examined.
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.
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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
During Dosing Period
Male: Salivation was observed in the vehicle control group (7112), 5 mg/kg group (616), 20 mg/kg group (4/6), 80 mg/kg group (416), 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 (616), 20 mg/kg group (4/6), 80 mg/kg group (516), and 320 mg/kg group (10/12). Staining of the hair (1112) 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.

BODY WEIGHTS (FIG,1, TABLE 2, ADDENDUM 2) 2.1 During Dosing Period
No abnormalities were noted in all groups.

During Recovery Period
No abnormalities were noted in all groups.

FOOD INTAKES
During Dosing Period
No abnormalities were noted in all groups.

During Recovery Period
No abnormalities were noted in all groups.

HEMATOLOGICAL EXAMINATIONS
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.

BLOOD CHEMICAL EXAMINATIONS
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
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.
Male: Increased white blood cells and casts in the urinary sediment were noted in the 320 mg/kg group.

ORGAN WEIGHTS
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.

NECROPSY
At Termination of Dosing Period
Male: Enlargement of the liver (116) was observed in the 80 mg/kg group. Pale change of the lungs (316), enlargement of the liver (516), and enlargement of the mediastinal lymph nodes (316) were observed in the 320 mg/kg group. Blackish region of the mucosa in the glandular stomach was observed in the vehicle control group (216), 5 mg/kg group (116), and 20 mg/kg group (116).
Female: Pale change of the lungs (116), enlargement of the liver (616), enlargement (1/6) and pale change (316) of the kidneys, and enlargement of the mediastinal lymph nodes (516) 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 (116) was observed in the 320 mg/kg group.

HISTOPATHOLOGICAL EXAMINATIONS
At Termination of Dosing Period
Male: Hypertrophy of the alveolar lining cells (+, 316 ; ++, 316) in the lungs, centrilobular hypertrophy (+, 116) and increased mitoses (+, 116) of the hepatocytes, focal myocarditis (+, 116), germinal center development (+, 116) and hypertrophy of the paracortex (+, 416) in the mediastinal lymph nodes were observed in the 320 mg/kg group. Necrosis of the fundic mucosa (+, 216) in the glandular stomach, focal myocarditis (±, 116), solitary cyst in medulla (+, 116) 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 (+, 111) and 20 mg/kg group (+, 111). Diffuse lipid droplets (++, 116) and focal necrosis (+, 116) 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 (+, 516) in the lung, centrilobular hypertrophy (±, 216 ; +, 316) of the hepatocytes, focal myocarditis (+, 316), atypical basophilic tubules (++, 6/6) and mineralization of the cortico-medullary junction (f, 116 ; +316) in the kidney, and hypertrophy of the paracortex (+, 416 ; ++, 116) in the mediastinal lymph node were observed in the 320 mg/kg group. Mineralization in the cortico-medullary junction (±, 116) and solitary cyst in medulla (+, 116) in the kidney were observed in the vehicle control group. Microgranuloma (+, 116) in the liver was observed in the 80 mg/kg group.

At Termination of Recovery Period
Male: Foamy cells (+, 516) and hypertrophy of the alveolar lining cells (+, 216) in the lung, focal myocarditis (+, 116), and hypertrophy of the paracortex (+, 116) in the mediastinal lymph node were observed in the 320 mg/kg group.
Female: Foamy cells (+, 616), and hypertrophy of the alveolar lining cells (+, 2/6) in the lung, atypical basophilic tubules (+, 316) and mineralization of the cortico-medullary junction (±, 116; +116) in the kidney, hypertrophy of the paracortex (+, 116) in the mediastinal lymph node, and increased extramedullary hematopoiesis (+, 216) in the spleen were observed in the 320 mg/kg group. Mineralization in the cortico-medullary junction (±, 216 ; +, 116) in the kidney was observed in the vehicle control group. Necrosis of the fluidic mucosa (+, I/1) in the glandular stomach, microgranuloma (+, 116) in the liver and solitary cyst (+, 116) in medulla in the kidney were observed in the 320 mg/kg group.

Effect levels

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed effect level (NOEL) of CC-5-V 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:

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 28-day repeated-dose toxicity study of orally administered RO-4562 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 CC-5-V 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.