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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:
1986-06-03 - 1987-02-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987
Reference Type:
other: Supplementary Report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted May 12, 1981
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Appearance: yellow liquid
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD (SD) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 4 - 5 weeks/period
- Weight at study initiation: 90-100 g
- Fasting period before study: no
- Housing: individually in Makrolon type III cages on soft wood bedding (LIGNOCEL (R), type 3/4
- Diet: ALTROMIN(R) standard diet No. 1324, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 15 -20
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: 03-June - 09-July 1986

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily as follows: 250, 1250 and 5000 mg of the test item were weighed into volumetric flasks and filled up with the carrier to a final volume of 50 mL each. Subsequently the compound suspensions in the volumetric flasks were kept homogeneous by placing on magnetic stirrers till the end of dosing.

VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Concentration in vehicle: 5, 25 or 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study, pretest samples from all dose levels of the test compound were analysed for stability, concentration and homogeneity. During the first week and the fourth week of the study samples from all dose levels were analysed for concentration and homogeneity. Concentration and homogeneity of all examined samples have been found to be acceptable. The test item has been proven stable.
Duration of treatment / exposure:
28 d
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were based on the results of an acute toxicity study and the dosages were establishedin accordance with the sponsor.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration; twice daily on 7 days/week. Observations for mortality/viability were recorded twice daily on 7 days/week.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded before treatment and daily at the time the animals were dosed.

FOOD CONSUMPTION / FOOD EFFICIENCY: Yes
Individual food consumption was recorded before treatment and once every week during treatment.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment and at the end of the treatment period all animals were subjected to an ophthalmologic examination. The eyes of the animals were examined with an ophthalmoscope and a slit-lamp. For exact examination of the lens, vitreous body and fundus of the eyes mydriasis was induced after installation of I % aqueous atropinesulfate into the conjunctival sac.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study; blood samples were collected between 7.00 and 9.00 a.m. (24 hours after compound administration) to prevent chronobiologic variations.
- Anaesthetic used for blood collection: no data; blood collection for coagulation parameter assessment: CO2
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell morphology, total leukocyte count, differential leukocyte count, platelet count, thromboplastin time, partial thromboplastin time, thrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the study between 7.00 and 9.00 a.m. (24 hours after compound administration) to prevent chronobiologic variations.
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: Glucose, urea, uric acid, creatinine, total bilirubin, total cholesterol, triglyceride, aspartate-aminotransferase, alanine-aminotransferase, lactate-dehydrogenase, gamma-glutamyl-transpeptidase, alkaline phosphatase, total protein (Biuret method and electrophoresis), inorganic phosphorus, chloride, sodium, potassium, calcium, albumin/globulin-ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalyses were done at pretreatment period, week 2 and, week 4 (urine collected in 3 hours, 24 hours after compound administration, initial water supply: 2 mL/100 g bw).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, color, turbidity, specific gravity, blood, leukocytes, nitrite, pH, protein, glucose, ketone, urobilinogen, bilirubin, sediment, sodium, potassium, calcium, chloride

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
At autopsy the following weights were recorded from all animals: adrenals, brain, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroid glands with parathyroid glands

HISTOPATHOLOGY: Yes
Microscopical examinations:
Adrenal glands, aorta, brain, caecum, colon, duodenum, epididymides, esophagus, eyes with optic nerves, femur, heart, ileum, pituitary gland, prostate, rectum, salivary gland (mandibular), sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (thoracic), spleen, sternum, stomach, jejunum, kidneys, liver, lungs with bronchi, lymph nodes (cervical/mesentery), mammary gland, ovaries, pancreas, testes, thymus, thyroid glands with parathyroid glands, tongue, trachea, urinary bladder, uterus, all gross lesions
Statistics:
Routine evaluation of the data was done by:
Student's t-test: Normal distributed values, single treatment groups were compared against the control group.
U-test: In not normal distributed values the U-test of Wilcoxon, Mann and Wliitney was employed to compare two autonomous random samples.
William's test: The control group was compared with treatment groups of constant increasing or decreasing dosage, to determine the lowest dosage, where a significant difference was apparent. The method used therefore was the Williams test (Trend test).
Bartlett's test: This test checks the homogeneity of variance in normal distributed data groups.
Global test: In biological datas mostly a sufficient normal distribution of values cannot be assumed. Therefore the H-test of Kruskal and Wallis was applied to compare several autonomous random samples.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed.
Mortality:
no mortality observed
Description (incidence):
No animal died during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A slight decrease in female rats and a slight increase in male rats were seen in relation to control, but no dose relation and no statistical significance were found.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In food consumption no remarkable differences were found. In females of group 50 mg/kg bw and 1000 mg/kg bw a slight statistical significant increase of mean food consumption in g/day/kg bw was observed at week 4, but no dose relation was seen.
Food efficiency:
no effects observed
Description (incidence and severity):
In food efficiencies no remarkable differences were found.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
At ophthalmologic examinations no abnormal signs or pathologic symptoms were found.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Differences of statistical significance between control and treated groups were found occasionally. All parameters were found to be in normal range and no dose relationship was seen, therefore the deviations were considered to be not of toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Differences of statistical significance between control and treated groups were found occasionally. All parameters were found to be in normal range and no dose relationship was seen, therefore the deviations were considered to be not of toxicological relevance.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In individual animals some parameters were observed slightly deviating from normal range, but no deviations related to the test substance were seen. Values slightly outside from normal range were found to be one time occurrences and therefore not considered to be compound related.
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):
No treatment related changes were observed. In males of group 50 mg/kg bw a statistical significant increase of the heart weight was found in relation to control (but only in absolute organ weight). In males of group 50 mg/kg bw a statistical significant decrease of the spleen was found (absolute and relative), but no dose relation was observed. In males of group 1000 mg/kg bw a statistical significant decrease of the thyroid glands was found (in relative organ weights). No dose relation was observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The main lesions observed at gross observation were claycolored or mottled livers and alterations of thymus. Most of the observed lesions were found to be histologically normal. Therefore these lesions were considered to be not compound related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No compound related lesions were found upon microscopic examinations of the tissues. The lesions most frequently seen included cytoplasmic vacuolation in the liver, hydronephrosis in the kidney, histiocytosis in the lungs, and posterior synechia in the eye. These lesions occurred with generally equal frequency in control and drug-treated groups, and therefore are considered not to be treatment-related. All other lesions observed occurred in one sex only, were one time occurrences and/or were seen in both control and treated groups and were therefore not considered to be treatment-related.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test-item related adverse effects up to and including the high dose level of 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The repeated administration of the test article by gavage for 28 consecutive days to rats at doses of 50, 250 and 1000 mg/kg did not result in compound-related effects. The No Observed Effect Level was found to be 1000 mg/kg body weight.
Executive summary:

In a 28-day repeated dose toxicity study according to OECD guideline 407 and in compliance with GLP, the test item in olive oil was administered daily to five rats/sex and dose level by gavage at dose levels of 0, 50, 250, and 1000 mg/kg body weight. Diet and water were provided ad libitum. All rats were observed at least twice daily for mortality and clinical signs of toxicity. Body weights were recorded daily, food consumption weekly. Ophthalmologic examinations were done during pretreatment and at week 4 of treatment. Hematology, blood chemistry evaluations and blood clotting investigations were conducted at termination of the study. Urinalyses were done during pretreatment and at week 2 and 4 of dosing. All rats were sacrificed after 4 weeks of treatment. Body weight of the exsanguinated animals and organ weights were taken and gross post mortem examinations were performed on all rats. A histopathological examination was done on all rats of the control and high-dose group and on all gross lesions seen at necropsy. No clinical signs of toxicity were observed and no animal died during the study. In body weight gain and food consumption no remarkable differences were seen between treated animals and control. At ophthalmologic examinations no abnormal signs or pathologic symptoms were found. In clinical laboratory investigations no deviations of toxicological relevance were seen, although differences of statistical significance between control and treated groups were found occasionally. The main lesions observed at gross observation were claycolored or mottled livers and alterations of thymus. The lesions are considered not to be compound related. No compound related lesions were found upon microscopic examinations of the tissues. As a result, the NO(A)EL was determined at 1000 mg/kg body weight in males/females.