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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guide line study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
neurobehavioural analysis was not performed
Qualifier:
according to
Guideline:
other: EEC Directive, Official Journal of the European Communities, 30.5.88, Sub-chronic oral toxicity test: 90-day repeated oral dose (rodent species).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: white, powder
- Analytical purity: 98.3 %
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: Tif: RAIf (SPF), hybrids of RII/1 x RII/2
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Production, CIBA-GEIGY Limited, 4332 Stein / Switzerland
- Age at study initiation: approximately 4-5 weeks at delivery
- Weight at study initiation: 111.0 - 129.9 g in males, 95.3 - 124.0 g in females
- Housing: groups of 5 in macrolon cages type 4 with wire mesh tops and standardised granulated soft wood bedding (Societe Parisienne des Sciures Pantin).
- Diet (e.g. ad libitum): Pelleted, certified standard diet (Nafag No. 890 Tox)
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: mixed with food and water
Details on oral exposure:
DIET PREPARATION
The tes substance was weighed on a calibrated Mettler balance. The pulverised food was then homogeneously mixed with the appropriate concentrations of the test article and about 25% water was added before pelleting to ensure the necessary pellet quality. The pellets were subsequently airdried. Diet was prepared at about monthly intervals and stored in stainless steel containers at room temperature in a separate area.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Control analyses of the test article content were undertaken with diet used for treatment days 1-29 and treatment days 57-94, respectively. These analyses were carried out in the analytical laboratories of Analytical Services Rosental, CIBA-GEIGY Limited 4002 Basle / Switzerland.
Duration of treatment / exposure:
92-93 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
150, 800, 3000, 15000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted range finder study (No. 884664)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (a.m. and p.m. on working days, a.m. on weekends and holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (midweek); the first weights were recorded during the acclimatisation period.

FOOD CONSUMPTION:
- Time schedule: weekly

FOOD CONSUMTION RATIO
- The food consumption ratios were calculated as mean of individual ratios. Unit: g food/kg bodyweight per day

WATER CONSUMPTION: Yes
- Time schedule for examinations: The water consumption was recorded weekly (cagewise).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to dosing (day -4) and towards the end (day 88) of the treatment period.
- Dose groups that were examined: animals of the control group and the highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period, in the morning
- Anaesthetic used for blood collection: Ether anesthesia
- Animals fasted: Yes, overnight
- How many animals: all surviving animals of each dose group
- Parameters checked: Erythrocyte Count, Hemoglobin, Hematocrit, Mean corpuscular weight, Mean corpuscular hemoglobin, Leucocyte Count, Differential Leucocyte, Count, Thrombocyte Count, Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period, in the morning
- Animals fasted: Yes, overnight
- How many animals: all surviving animals of each dose group
- Parameters checked: Glucose, Urea, Creatinine, Total bilirubin, Total protein, Albumin, Globulins, A/G Ratio, Cholesterol, Sodium, Potassium, Calcium, Chloride, inorganic Phosphorus, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl transpeptidase

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period, overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Food and water was withheld during the time of urine collection.
- Parameters checked: Urine volume, Urine relative densitiy, pH-value, Protein, Glucose, Ketones, Bilirubin, Blood, Urobilinogen; pH, Protein, Glucose, Ketones, Blood, Bilirubin and Urobilinogen were estimated by an automated urine chemistry analyser Clinitek Auto 2000 (Ames), using solid-phase reagent systems and reflectance spectroscopy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Besides the weight of the exsanguinated body the following organs were weighed: brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen

HISTOPATHOLOGY: Yes
The following organs and tissues were preserved in neutral buffered 4% formalin:
skin
mammary area
spleen
mesenteric lymph node
axillary lymph node
popliteal lymph node
sternum with bone marrow
femur with joint
skeletal muscle
trachea
lung
heart
aorta
submandibular salivary gland, both
liver
pancreas
esophagus
stomach
small intestine
large intestine
kidney, both
urinary bladder
prostate
seminal vesicle
testis, both
epididymis, both
uterus
vagina
ovary, both
pituitary gland
adrenal gland, both
thyroid with parathyroid gland
thymus
peripheral nerve
brain
spinal cord
eye with optic nerve, both
orbital gland, both
extraorbital lacrimal gland, both
Zymbal gland, both
muzzle
tongue
any tissue with gross lesions

After the fixation, organ samples listed above except popliteal lymph node, zymbals gland and muzzle, were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination.
Statistics:
For each time point and parameter an univariate statistical analysis is performed. Nonparametric methods are applied, to allow for non normal as well as normal data distribution.
Each treated group is compared to the control group by Lepage's two-sample test. This test is a combination of Wilcoxon and Ansari-Bradley statistics, i. e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion.
Increasing or decreasing trends in location from control to the highest dose group are tested by Jonckheere's test for ordered alternatives. This test is sensitive to monotone dose-related treatment effects.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No relevant clinical symptoms and no signs of systemic toxicity were observed during the study. Except for one female rat (No. 56) of the control group which was found dead on study day 72, no death occurred during the study.

BODY WEIGHT AND WEIGHT GAIN
The mean bodyweight gain of all treated groups was considered undisturbed and comparable to the respective controls. A few values reaching statistical significance were considered unrelated to the treatment and of no biological relevance.

FOOD CONSUMPTION
The mean food consumption of male group 5 (15000 ppm) was considered slightly increased from week 5 onwards, reaching a final value of 13% above the control. In the absence of a dose-relationship, the mean food consumption of all other treated groups were considered within the normal range and undisturbed by the treatment.
Compared to the control group, the mean focxi consumption ratios of male group 5 (15000 ppm) were slightly increasing from week 3 onwards until the end of the study, reaching a final value of 12.2% above the control. The mean food consumption ratios of all other treated groups were comparable to the respective controls.

WATER CONSUMPTION
In the absence of a dose-relationship, the mean water consumption of all treated groups was considered undisturbed by the treatment. Minor fluctuation may reflect biological variability without toxicological relevance.

OPHTHALMOSCOPIC EXAMINATION
Examinations included inspection of the surroundings of the eyes, of sclera, cornea, iris and adaptation of the pupil to the ophthalmoscopic light beam. Animals of control and highest treatment level were examined at beginning (day -4) and towards the end (day 88) of the application period. The examinations revealed no evidence of a reaction to the treatment.

HAEMATOLOGY
Minimally higher numbers of blood platelets were observed in females of groups 4 and 5 (3000 and 15000 ppm). In the absence of corroborative findings these minor elevations of circulating platelets were not considered to be an adverse effect but might result from a stimulation of megakaryocytopoiesis.

CLINICAL CHEMISTRY
There were some parameters reaching a level of statistical significance in the difference between treated and control groups. However, all these minor differences represent the normal physiological fluctuation of the respective parameters and were therefore not considered treatment-related. No experimental relevance is attributed to decreased plasma urea levels as have been observed in the males of group 3, 4 and 5.

URINALYSIS
The values for treated and control groups were comparable and no treatment-related changes were noted.

ORGAN WEIGHTS
Statistical analysis of both absolute organ weights and organ to bodyweight ratios did not reveal any treatment-related effects. A few statistically significant differences in absolute and relative organ weights were considered unrelated to the treatment and of no toxicological relevance.

GROSS PATHOLOGY
All findings occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance is attributed to these findings.

HISTOPATHOLOGY
A variety of different non-neoplastic lesions found in this study commonly occur in our colony of rats. Neither their incidence nor their nature gave any indication of a treatment related effect. The haemorrhages and/or inflammatory cell infiltrations in the harderian gland and/or the periocular tissue observed in all animals used for blood collection are considered to be artefacts and are not reported.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
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: Based on the analytically found value in the diet
Dose descriptor:
NOEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: slight increase of food consumption and consumption ratios in group 5 males (15000 ppm)
Dose descriptor:
NOEL
Effect level:
800 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: elevated platelet counts in females treated at 3000 and 15000 ppm

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL > 1000 mg/kg bw/day