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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 March 18 to April 29, 1991.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted May 12, 1981
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fuellinsdorf.
- Age at study initiation: 8-9 weeks.
- Weight at study initiation: males: 164 - 176 g; females: 143 - 160 g.
- Housing: individually in Makrolon type-3 cages (size: 22 x 37.5 x 15 cm) with autoclaved standard softwood bedding ('Lignocel', Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Kliba no. 343, Batch 81/91 and 83/91 rat maintenance diet ('Kliba', Klingentalmuehle AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap-water, ad libitum.
- Acclimation period: seven days under laboratory conditions, after veterinary examination.


ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40-70 %
- Air changes: 10-15 air change/haour
- Photoperiod: 12 hours light cycle day.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Bi-distilled water
Details on oral exposure:
PREPARATION
- Test article preparation: the test article was weighed into a glass beaker on a tared Mettler PM 480 balance and the vehicle, bi-distilled water, were added. The mixtures (w/v) were prepared using a homogenizer. Homogeneity of the vehicle was maintained during treatment using a magnetic stirrer.
- Frequency of preparation: daily prior to each application.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the mixtures were determined during acclimatization. Further samples for analysis were taken during week 3 of the test and subsequently analyzed.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week for 28 days.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 41, 165 and 825 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
0 and 100 mg/kg bw: 10 x sex x dose
50 and 200 mg/kg bw: 5 x sex x dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: results from a 5-day range-finding study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily.

DETAILED CLINICAL OBSERVATIONS
Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT
The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PM 4800 balance connected to the laboratory computer.

OPHTHALMOSCOPIC EXAMINATION
At the 4th and 6th week; ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at termination of treatment and a second time on the recovery individuals of groups 1 and 4 at termination of the recovery period. Approximately 10-90 minutes after the application of a mydriatic solution the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Miroflex 2 Ophthalmoscope. Pictures were taken of all abnormal findings.

HAEMATOLOGY
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected between the hours of 07.05 and 08.35 to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Nucleated erythrocytes, Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red cell morphology, Thromboplastin time.

CLINICAL CHEMISTRY
Parameters examined: Glucose, Urea, Creatinine, Uric acid, Bilirubin, total Cholesterol, total Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, Protein total, Albumin, Globulin, Albumin/Globulin ratio.

URINALYSIS
Urine was collected during the 18-hours fasting period into a specimen vial, using a metabolism cage.
Parameters examined: Volume, Specific gravity, Color, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Urobilinogen, Urine Sediment.
Sacrifice and pathology:
ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were taken from all animals necropsied at termination of treatment or recovery: Adrenals; Brain; Heart; Kidneys; Liver; Ovaries; Pituitary gland; Spleen; Testes; Thyroid gland.

NECROPSY
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. Prior to necropsy, the animals were fasted for approximately 18 hours, but water was provided. Necropsies were performed by experienced prosectors.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in phosphate buffered neutral 4% formaldehyde solution: Adrenals; Aorta; Bone (sternum, femur); Bone marrow (sternum, femur); Brain; Cecum; Colon; Duodenum; Epididymides; Esophagus; Eyes with optic nerve and Harderian gland; Female mammary gland area; Femur including joint; Heart; Ileum; Jejunum; Kidneys; Larynx; Lacrimal gland, exorbital; Liver; Lung infused with formalin; Lymph nodes, mandibular, mesenteric; Nasal cavities; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary gland, mandibular, sublingual; Sciatic nerve; Seminal vesicles; Skeletal muscle; Skin; Spinal cord, cervical, midthoracic, lumbar; Spleen; Stomach; Testes; Thymus; Thyroid gland; Tongue; Trachea; Urinary bladder infused with formalin; Uterus; Vagina; Gross lesions.

HISTOPATHOLOGY
All organ and tissue samples, as defined under Histopathology were processed, embedded, cut at a thickness of 2-4 micrometers, and stained with hematoxylin and eosin. Slides of Adrenals, Heart, Kidneys, Liver, Spleen and Stomach collected at terminal sacrifice from the animals of the control and high-dose groups were examined by a pathologist. The same applied to all gross lesions.
Statistics:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. Group means were calculated for continuous data and medians were calculated for discrete data (scores).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no statistically or toxicologically significant effect
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no toxicologically relevant significant effects
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis 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
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related clinical findings were observed during the study period nor during the treatment-free recovery period. No premature death occurred before scheduled time of necropsy.

BODY WEIGHT AND WEIGHT GAIN
The body weights of all treated animals were comparable to each other. Therefore, it was considered that the treatment with test substance showed no toxicologically relevant effects on the body weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Females of group 4 (1000 mg/kg bw ) ate statistically significantly less food than controls from treatment days 8 to 15 and male rats of the same group consumed statistically significantly more food than controls from treatment day 22 until 28 (end of the treatment period). The relative food consumption rates of male rats of groups 3 and 4 (200- and 1000 mg/kg) were significantly higher from treatment day 22 until 28 when compared to those of controls. These findings were considered to be toxicologically not relevant and within normal biological variations known for these animal-strain and age.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examination of each animal towards the end of the treatment period and each recovery animal after additional 14-days of treatment-free recovery period did not reveal any clinical findings.

HAEMATOLOGY
The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. All differences in the results of analysis parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

CLINICAL CHEMISTRY
The assessment of clinical biochemical data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. All differences in the results of analysis parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

URINALYSIS
The assessment of urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period. All differences in the results of analysis parameters were considered to be incidental and of normal biological variation for rats of this strain and age.

ORGAN WEIGHTS, ORGAN TO BODY WEIGHTS AND ORGAN TO BRAIN WEIGHT RATIOS
At termination of the 4-week treatment period, the kidneys to brain weight ratios of males of groups 2 and 4 (50- and 1000 mg/kg bw) were statistically significantly higher and the heart to brain weight ratios of females of group 4 (1000 mg/kg bw) statistically significantly lower than those of the controls. This was considered to be toxicologically not relevant and therefore incidental as there were no confirmatory findings in the other sex, nor confirmatory macroscopical or microscopical findings. No other significant differences from controls occurred in absolute and relative organ weights.

MACROSCOPIC AND MICROSCOPIC FINDINGS
No treatment related macroscopic or microscopic findings were recorded. The small number of findings recorded are within the normal range of background alterations which may be seen in untreated animals of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related microscopic findings were recorded.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
825 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on the active substance content of 82.5 %
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (28 days): 825 mg/kg bw/d (based on the active substance content of 82.5 %) for male and female rats.
Executive summary:

Method

A subacute 28-day toxicity study was performed administering the test substance daily by gavage to SPF-bred Wistar rats. The study was comprised of four groups dosed at 0, 20, 200 and 1000 mg/kg.

Results

No premature death occurred.

There were no toxicologically relevant significant effects on absolute or relative food consumption, on the body weight in the test article treated animals when the results were compared to those of the controls.

No clinical signs were observed during the entire study period. No clinical abnormalities were found on ophthalmoscopy.

The assessment of hematology, clinical biochemical and urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.

No changes in absolute and relative organ weights were observed after 4 weeks of treatment nor at termination of the treatment-free recovery period when the results from the animals of the test article treated groups were compared to those of the control animals.

No treatment related findings were recorded at termination after 4 weeks of treatment nor at termination of the treatment-free recovery period.

Conclusion

Based on the results obtained the "no-adverse-effect-level" of the test substance is 1000 mg/kg body weight for male and female rats when administered orally by gavage for a period of 28 days followed by a treatment-free 14-day recovery period.

The various statistically significant differences observed (increase/decrease) in food consumption (absolute/relative) and organ weights (relative) were considered to be of no toxicological relevance and did therefore not provide any evidence of toxicity at any of the dose levels tested.