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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 November to 21 December 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in compliance with GLP regulations and method followed was comparable to OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The method followed was broadly comparable with current OECD guidelines. The test substance, formulated as a solution in corn oil was administered to rats by intragastric intubation at dosage levels of 10, 100 or 1000 mg/kg/day. Treatment was carried out once daily for twenty-eight consecutive days. Control animals similarly received corn oil (5 mL/kg/day).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: Not specified
Batch No.: Not specified

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage, Michigan, USA.
- Age at study initiation: 28 ± 1 days old.
- Weight at study initiation: 75 to 94 g.
- Fasting period before study:
- Housing: Initially caged, in groups of five according to sex.
- Diet (e.g. ad libitum): Free access to Labsure LAD 1 Diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: An 11-day acclimation period was allowed between delivery of the animals and start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1℃ to 19.4℃
- Humidity (%): 47.5% to 55.7%
- Air changes (per hr): Approx. 19 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light in each 24-hour period.

IN-LIFE DATES: From: 10 November 1988 To: 19 December 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Preparation of dosing solutions:
The high dosage concentration of the test substance was prepared freshly each day as a 20% w/v solution in corn oil. The intermediate (2% w/v) and low (0.2% w/v) dosage concentrations were similarly prepared by direct dilution of the test substance with the vehicle.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration analysis of solutions prepared for administration on Day 1 were analysed by GC.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Animals were treated once daily, seven days per week for four weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 100 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dosage levels of the test substance were selected on the basis of acute oral toxicity data available from the sponsor and a 7-day preliminary oral rat toxicity study carried out at HRC.
The test substance was administered by oral gavage to rats of groups 2 to 4 inclusive using a syringe and rubber catheter at a dosage volume of 5 mL/kg/day.
Control animals similarly received corn oil (5 mL/kg/day).
Each animal received a constant dosage level based on its most recently recorded bodyweight.
Animals were treated once daily, seven days per week for four weeks.
Positive control:
None stated

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.
All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. This allowed a post mortem examination to be undertaken during the working part of that day. At weekends a similar procedure was followed except that the final check was carried out at mid-day.
Rat No. 22, which was found dead on Day 14 was subjected to a detailed macroscopic examination in an attempt to define the cause of death. Any abnormal tissues and all tissues listed under Terminal studies were subjected to histological examination.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

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
The quantity of food consumed in each cage was measured at weekly intervals throughout the study.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was withdrawn under light ether anaesthesia from the orbital sinus of all surviving rats prior to termination (week 4).
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, food was withdrawn overnight prior to collection of samples.
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was withdrawn under light ether anaesthesia from the orbital sinus of all surviving rats prior to termination (week 4).
- Animals fasted: Yes, food was withdrawn overnight prior to collection of samples.
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

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

OTHER:
Microscopic examination:
Fixed tissue samples required for microscopic examination were embedded in paraffin wax, sections cut at 4 μm and stained with haematoxylin and eosin.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, adrenals, liver, testes (with epididymides), kidneys and ovaries.
HISTOPATHOLOGY: Yes, adrenals, kidneys, spleen, heart, liver and any other macroscopically abnormal tissue.
Other examinations:
None stated
Statistics:
The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology data:
(1) If the data consisted predomonantly of one particular value, the proportion of values defferent from the mode was analysed by appropriate methods. Otherwise:
(2) Bartlett's test was applied to test for heterogeneity of variance between treatments.
(3) If no significant heterogeneity was detected, a one-way analysis of variance was carried out. If significant heterogeneity of variance was present, and could not be removed by a transformation, the Kruskal-Wallis analysis of ranks was used.
(4) Analyses of variance were followed by Student's ‘t’ test and Williams' test for a dose-related response, although only the one thought more appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of the 't' test and Williams' test.
For organ weight data, where appropriate, analysis of covariance was used in place of analysis of variance in the above sequence. The final bodyweight was used as covariate in an attempt to allow for differences in bodyweight which may have influenced the organ weights.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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 specified
Details on results:
Clinical findings:
Hunched posture and increased salivation following dosing were noted during the study period amongst rats in all dosage groups receiving the test substance.
Abnormal gait (waddling), lethargy, noisy respiration and a thin appearance were also noted amongst female rats in the intermediate dosage group and amongst both male and female rats in the high dosage group.

Bodyweight changes:
In comparison with controls, significantly lower bodyweight gains were recorded during weeks 1, 3 and 4 for male rats receiving the test substance, 1000 mg/kg/day.
Bodyweight gains for rats in all other dosage groups were similar to those of the controls.

Food consumption:
Food consumption for male rats in the high dosage group was slightly lower than that of the controls during the four-week study period. These changes in food consumption can be related to the lower bodyweight gains of male rats receiving the test substance, 1000 mg/kg/day. Food consumption for female rats in the high dosage group was slightly higher than that of the controls.

Haematology:
No changes in haematological parameters were noted that were considered to be treatment-related.

Biochemistry:
Significantly higher glutamic-pyruvic transaminase levels were recorded during week 4 for both male and female rats in the high dosage group in comparison with those receiving corn oil. A similar, but not statistically significant, trend to higher glutamic-oxaloacetic transaminase levels was recorded for rats receiving the test substance, 1000 mg/kg/day were also signigicantly higher than those of the controls.

Organ weight analysis:
Adjusted liver weights for female rats receiving the test substance, 1000 mg/kg/day were significantly higher than those of the controls following the four-week treatment period. No significant changes in liver weights were recorded for male rats in the high dosage group.

Macroscopic pathology:
Badly groomed fur, dirty tails and brown staining of the pinnae were observed at termination amongst rats receiving the test substance, 1000 mg/kg/day.

Microscopic pathology:
Minimal centrilobular hepatocyte enlargement was noted in the liver of two male and two female rats in the high dosage group. This liver change, which was considered to be treatment-related, was not observed for rats in the lower dosage groups.

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No findings of toxicological significance at 100 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Changes in general health, bodyweight gains, food consumption, biochemical parameters, organ weights, macroscopic and microscopic pathology were recorded for rats in the high dosage group receiving the test substance, 1000 mg/kg/day.
Several abnormal clinical findings were also noted for rats in the lower dosage groups. However, in the absence of any other physiological or morphological changes these changes were not considered to be of major toxicological importance. The level of the test substance at which no overt signs of toxicity were noted was therefore considered to be 100 mg/kg/day.