Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June - Nov 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: 28-day Repeated Dose Toxicity Study in Mammalian Species (Notification No. 1121002, Manufacturing Industries Bureau METI & No. 033121002 of the Environmental Health Department, MOE)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Chalres River Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: 140.3 - 163.2 g (males), 117.4 - 143.9 g (females)
- Housing: in hanging stainless steel cages with wire mesh floor at 5 animals/cage for acclimation; at 1 animal/cage for study
- Diet (e.g. ad libitum): MF pelleted diet (lot nos. 060306 and 060509, Oriental Yeast), ad libitum
- Water (e.g. ad libitum): chlorinated water from Hita City supply, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.4-24.3°C
- Humidity (%): 48.3-65.0%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27. June To: 17. Aug. 2006

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):
When the preparation of the dosing formulation was investigated, the suspended state in the olive oil was good. Therefore, olive oil was selected as the vehicle
- Concentration in vehicle:
The substance was accurately weighed and suspended in an agate motor to prepare 10.0. w/v% formulation. The lower concentrations of 1.5 and 0.25 w/v% fomulations were diluted with olive oil from 10.0 w/v% formulation. These were prepared once weekly because 10.0 and 0.5 w/v% formulations were stable for 7 days.
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 038OHS (Fujimi Pharmaceutical)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the start (first preparation) of dosing, middle layers of 10.0, 1.5 and 0.25 w/v% formulations were taken just after preparation, and quantitatively analyzed by HPLC after samples pretreatment. The rate to the nominal concentration for the actual concentration of all formulations were within the range of 100 +/- 10%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
25 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a 7 day pre study (50, 250, 500 and 1000 mg/kg bw/d)
- Post-exposure recovery period in satellite groups: 14 days (0 and 1000 mg/kg bw/d)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day (amin study); twice a day (recovery period)
general condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing and once weekly thereafter
1. Observation in arena
Animals were placed in the standard arena and observed, and defecation and urination were recorded for 1 min. Posture, motor activity level, respiration, lid closure, gait characteristics, tremor, twitch, convulsion, stereotypes, abnormal behavior
2. Removal from cage
The case with which the animal was removed from the home cage was recorded.
Ease of removal, vocalisation
3. Handling observations
In the hand observations
muscle tone, subnormal temperature, piloerection, hair appearance (staining, unkempt hair), skin color (paleness, reddening, cyanosis), eyey (lacrimation, exophthalmos, pupillary size), salivation, secretion

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was measured on day -2 before initiation of dosing, on days 1, 3, 8, 12, 17, 21, 26 and 28 during the dosing period, and on days 1, 5, 10 and 14 (recovery) during the recovery period. In addition, immediately before necropsy, body weights were measured for calculation of relative organ weights

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured once before initiation of dosing, on days 3, 8, 15, 22 and 28 during the dosing period, and on days 4, 8 and 14 (recovery) during the recovery period.

HAEMATOLOGY: Yes
Blood or plasma samples obtained by blood sampling from the abdominal aorta under ether anesthesia after overnight fasting (16 to 20 hr) at completion of the dosing period (excluding the recovery groups) and at completion of the recovery period were determined for the following items: red blood cell count, white blood cell count, hemoglobin concentration, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, prothrombin, activated partial thromboplastin time, differentiation of leukocytes (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells)

CLINICAL CHEMISTRY: Yes
Serum samples were separated from blood samples collected at the same time as those described in Hematology. The following items were determied in the serum samples:
Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Cholinesterase (ChE), gamma-Glutamyl transpeptidase, total Cholesterol, Triglyceride, Glucose, total protein, Albumin, A/G ratio, blood Urea nitrogen, Creatinine, total Bilirubin, Calcium (Ca), inorganic Phosphorus (IP), Sodium (Na), Poassium (K) and Chloride (Cl)

URINALYSIS: Yes
Urinalyses for the following items were performed once (day 28) during the dosing period (excluding recovery groups) and once (day 14 (recovery)) during the recovery period. Urine samples were collected (accumulated fro 15-17 hr) with individual metabolic cages under fasting, but drinking water ad libitum. The irinary sediments were stained and examined in males and females of the vehicle control and 1000 mg/kg bw/d groups at the end of the dosing period:
Urine volume, color, turbidity, urine specific gravity, pH, protein, Glucose, occult blood, urinary sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
All animals were examined in week 4. The detailed examinations were followed by the reflex test. The observation in the recovery period was not conducted since no abnormalities were noted in week 4 of the dosing period.
1. Reflex
Sensory reactivity to stimuli of different types was performed ad blind tests after detailed clinical observation.
(1) Approach contact/touch response: A blunt probe was bought approx. 3 cm from the animal´s nose for 4 seconds.
(2) Pinna response: The animal´s response to a sudden sound of finger snap was assessed.
(3) Pain response: The animal´s tail was pinched with a clothspin between one-third and base of the tail.
(4) Pupillary reflex: Following darkness adaptation of the animal´s eyes, pupil constriction in response to a bright beam of light was observed.
(5) Air righting reflex: The animal was held ventra surface uppermost approx. 30 cm height from the flat surface and released.

2. Grip strength
The forelimbs and hindlimbs grip strengths were measured with automated grip strength meter ad blind tests. Two trials were performed and the mean values were calculated for each animal.

3. Locomotor activity counts
Locomotor activity level of each animal was counted with animal activity monitoring system by the number of crossing IR beam for 1 hour at 10 min. intervals.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
1. Necropsy
All animals were subjected to the detailed gross necropsy including body surface, all orifices, cranial, thoracic and abdominal cavities.
2. Organ weights
The weights of the following organs were measured in all animals:
Liver, heart, kidneys, testes, epididymides, ovaries, brain, spleen, thymus and adrenals.
The relative organ weight was also calculated based on the body weight at the time of necropsy.

HISTOPATHOLOGY: Yes
The following organs and tissues were taken:
Trachea, lung, stomach, intestine (duodenum to rectumwith Peyer´s patches), liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicle, ovaries, uterus, vagina, brain (cerebrum, cerebellum and pons), spinal cord, sciatic nerve, bone marow (femur), axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids (with parathyroids), adrenals, eye ball
Light microscopic examinations were performed for the following organs and tissues after embedding in paraffin, secioning and hematoxylin staining:
Trachea, lung, forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, bone marrow, axillar and mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroids, parathyroids, adrenals, eyeball
Statistics:
Data regarding body weights (excluding those at the time of necropsy), food intakes, hematological examinations, blood chemical examinations, urine volume and specific gravity, organ weights, grip strength and locomotor activity count 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, 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.

Defecation and urination were analyzed using the Kruskal-Wallis´s test. 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 nonparamtric Duenna´s test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
- Hematological Examinations:
a. At termination of dosing period
Males: White blood cell count decreased in the 25 mg/kg bw/d group. Eosinophil count increased in the 150 and 1000 mg/kg bw/d groups.
Females: No abnormalities

b. At termination of recovery period
Males: Decreased red blood cell cout and increased mena corpuscular volume and mean corpuscular hemoglobin in the 1000 mg/kg bw/d group.
Females: No abnomalities.

- Blood chemical Examinations:
a. At termination of dosing period
Males: T-Protein was decreased in the 1000 mg/kg bw/d group.
Females: Inorganic phosphorus was increased in the 1000 mg/kg bw/d group.

b. At termination of recovery period
Males: Potassium was increased in the 1000 mg/kg bw/d group.
Females: Blood urea nitrogen was increased in the 1000 mg/kg bw/d group.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOEL and the NOAEL of the test substance in rats under the present study conditions were estimated to be 1000 mg/kg/day.
Executive summary:

A 28 -day repeated oral dose toxicity study of the test item followed by a 14 -day recovery study was performed in groups of five male and five female Crl:CD (SD) rats at 5 weeks of age. The high dose was set at 1000 mg/kg/day, and altogether 3 doses including 150 and 25 mg/kg/day were employed. Recovery groups were also set for the 1000 mg/kg and vehicle control groups.

No death occurred and no abnormalities were noted in all examinations.

No abnormalities were noted in the recovery test.

Based on these results, the NOEL and NOAEL of the test item in rats under the present study conditions were estimated to be 1000 mg/kg/day.