Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 July 1994 to 14 November 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Japanese Guidelines on Industrial Chemicals (1986)
Deviations:
yes
Remarks:
No FOB was performed, some recommended organs were not weighted and/or examined.
GLP compliance:
yes
Remarks:
Compliant with Japanese GLP Standards
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

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
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5 weeks old
- Weight at study initiation: Males: 142 - 175 g and Females: 116 - 144 g
Housing: Two rats of the same sex were housed in a single polycarbonate cage (256 mm W x 426 mm D x 200 mmH) from Tokiwa Kagaku Kikai Co., Ltd
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:
A pellet diet (MF Oriental Yeast Co., Ltd) was provided and changes once a week. Ordinary tap water was filtered through a 5 - µm filter and disinfected by UV irradiation before being provided ad libitum to animals. Drinking water bottles were changed once a week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25- Humidity (%): 40 - 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test substance was administered orally using a gastric tube
Vehicle:
water
Remarks:
pure water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solution for the highest-dose group was prepared by dissolving the test substance in pure water. The dosing solutions for mid-dose and low-dose groups were prepared by diluting the higher-dose solution with pure water. The frequency of preparation was daily until 5-day stability was confirmed, once every 5 days until 10-day stability was confirmed, and once a week thereafter. The dosing solutions were stored in a refrigerator with tight containers.

DIET PREPARATION
- Rate of preparation of diet (frequency): once a week
- Storage temperature of food: Not specified

VEHICLE
- Concentration in vehicle: 1000 mg/kg, 140 mg/kg and 20 mg/kg
- Amount of vehicle (if gavage): dosing volume was 1.0 ml / 100 g bw (based on the most recent weight)
- Purity: pure water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
The test substance was administered to each rat, every morning, once a day for 28 days.
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
140 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 animals / sex / dose
6 recovery animals / sex for the highest dose and the control
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A preliminary 7-day study was carried out with doses of 500 and 1000 mg/kg bw with 3 male rats and 3 female rats per group. No abnormalities were observed.
- Rationale for animal assignment (if not random): Rats were grouped by the 'stratified-by-weight' randomised method

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed daily for evidence of any systemic reactions to treatment or any other illnesses by assessing clinical signs and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed on the first day of treatment, and then once a week thereafter with an electronic balance

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the inferior vena cava of non-fasting rats under anesthesia at the scheduled sacrifice
- Anaesthetic used for blood collection: Yes (intraperitoneal thiopental sodium anesthesia - Ravonal, Tanabe Seiyaku Co., Ltd)
- Animals fasted: No
- How many animals: Not specified

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Same an haemotology tests occurred above
- Animals fasted: No
- How many animals: Not specified

URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine was collected from all rats at 26 days after the beginning of the treatment.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
Bartlett's test was used first, then groups were compared by analysis of variance when group variance was determined to be homogeneous. Dunnett's multiple range test or Scheffé's test was used when intergroup differences were significant. Comparisons were done by the Kruskal-Wallis test when Bartlett's test indicated heterogeneous group variance. When intergroup differences were significant, Dunnett's rank sum test or Scheffé's test was used. Urinalysis data was analysed by Armitage's x(2) test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No changes were noted in any animals during the treatment or recovery periods
Mortality:
no mortality observed
Description (incidence):
No animals died during the treatment and recovery periods
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight of all treated groups showed a similar increase to the body weight of the control group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The control group and treated group had a similar food consumption
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes due to the test substance was detected in either the treated or recovery periods. At the end of the treatment period, the hematocrit increased in male rats of the 140 mg/kg group, this was not found in the 1000 mg/kg therefore was not ascribed to the test substance. At the end of recovery period the reticulocyte count was increased in females and the MCHC was increased in males, the percentage of lymphocytes in the WBC count was decreased in females. However these changes were not due to the test substance, because they were slight and not found at the end of the treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, the glucose level was increased and the A/G ratio was decreased in female rats exposed to 140 mg/kg. These changes were not ascribed to the test substance because no changes were observed in the 1000 mg/kg group. At the end of the recovery period, glucose levels was increased in males, however this was not due to the test substance because it was not found at the end of the treatment period.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The level of ketones was increased in female rats exposed to 20 mg/kg. This change was not due to the test substance, because it was not found in the group exposed to 140 mg/kg or 1000 mg/kg exposed groups.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, the absolute ovarian weight was decreased and the relative brain weight was increased in female test subjects exposed to 1000 mg/kg. These changes were slight and were considered to be within the normal range. At the end of the recovery period, the total adrenal weight in males was decreased and the absolute and relative testicular weights were increased. However, these changes were not associated with the test substance as no such changes were found at the end of the treatment period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, thickening of the right femur was found in one control male and hepatodiaphragmatic nodule was found in one control female. These changes were considered to be incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Basophilic change of the renal tubular epithelium and cystic dilation of the renal tubules, focal necrosis, and focal myocardial degeneration were found in rats of the control and groups exposed to 1000 mg/kg. However, these lesions were considered to be incidental findings or spontaneous changes as they showed a similar incidence in the two groups. The thickened femur of the control male showed no histological abnormalities.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Hepatic microgranuloma were found in rats of the control and groups exposed to 1000 mg/kg. However, these lesions were considered to be incidental findings or spontaneous changes as they showed a similar incidence in the two groups.
Other effects:
not examined
Details on results:
Body weight, food consumption, hematology tests, clinical chemistry tests, urinalysis and pathological examinations revealed no evidence of toxicity.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

See attached report for all graphs and tables.

Applicant's summary and conclusion

Conclusions:
The NOAEL for the test substance was found to be greater than 1,000 mg/kg bw/day in the test animals. No significant adverse effects were observed.
Executive summary:

A 28 -day repeat dose toxicity study was performed on the registered substance using a method similar to OECD Test Guideline 407 (GLP) with deviations. The test substance was administered orally at doses of 20, 140 and 1000 mg/kg bw/day to Sprague-Dawley rats. Pure water was used as a vehicle and this was also used in the control group. Recovery animals were included for the control and highest dose, and observed for 14 days after the end of the treatment.

Animals were observed daily for clinical signs. Body weights were recorded on the first day of the treatment then weekly. Haematology and clinical chemistry were investigated, and urinalysis performed. At the end of the treatment or of the recovery period, animals were sacrificed, and a gross pathology and histopathology performed. All the rats survived to the end of the studies. No clinical signs or effects on body weight were observed in any of the rats. Changes were detected in the haematology and clinical chemistry parameters, and during the urinalysis, organ weighting, and histopathology, but they were not considered as significant and treatment-related.

It is concluded that no significant signs of toxicity were observed. The NOAEL was determined to be 1,000 mg/kg bw/day as it was the highest dose evaluated.