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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
27 July 1995
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanIbm:WIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Fuellinsdorf / Switzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 131 - 162 grams (mean 148 grams), Females: 105 - 143 grams (mean 119 grams)
- Housing: groups of five animals
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 (batch no. 40/99 and 41/99) rat maintenance diet (Provimi Kliba AG, Kaiseraugst/ Switzerland) ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/ 12 hours dark (light period between 06.00 and 18.00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article formulations were prepared weekly.
The test substance was weighed into a glass beaker on a tared Mettler balance and a small amount of vehicle added. After stirring with a glass rod the vehicle was added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C).
Homogeneity of the test article in the vehicle were maintained during the daily administration period using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For analytical verification the high performance liquid chromatography was carried out. This chemical analysis determined concentration, homogeneity, and stability of the test substance in PEG 300. The mean concentrations of the homogeneity samples were found to be 95.0%, 106.0%, and 102.7% of the nominal concentrations of the three dose groups 10, 40 and 200 mg/ml. The individual concentrations varied in the range from -8% to +4% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.
Duration of treatment / exposure:
28 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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:
- control: 10
- 50 mg/kg bw: 5
- 200 mg/kg bw:5
- 1000 mg/kg bw: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: test for reversibility of effects or late developing effects
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no

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 days 1-3 (ca 1 and 3 hours after administration); as well as once daily on days 4-28 (ca. 1 hour after administration) and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION:
- The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

HAEMATOLOGY: Yes
- Parameters checked: Erythrocyte count; Hemoglobin; Hematocrit; Mean corpuscular volume; Mean corpuscular hemoglobin; Mean corpuscular hemoglobin concentration; Platelet count; Reticulocyte count; Reticulocyte fluorescence ratios; Nucleated erythrocytes (normoblasts); Heinz bodies; Methemoglobin; Total leukocyte count; Differential leukocyte count; Red blood cell morphology; Thromboplastin time (=prothrombin time); Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- Animals fasted: Yes
- Parameters checked: 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; Albumin; Protein, total; Globulin; Albumin/Globulin ratio

URINALYSIS: Yes
- Parameters checked: Volume (18-hour); Specific gravity; Osmolality; Color; Appearance; pH; Protein; Glucose; Ketone; Bilirubin; Blood; Nitrite; Urobilinogen; Urine sediment; Red blood cells; Crystals (Triple phosphate)

MORTALITY/VIABILITY: Yes
- Time schedule for examinations: Observations for mortality/viability were recorded twice daily
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Student's T-Test was applied to locomotor activity and grip strength.
- Fisher's exact-test was applied to macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No test article-related clinical signs were evident during daily observations.
Mortality:
no mortality observed
Description (incidence):
One male (no. 25) treated at 1000 mg/kg/day died accidentally after blood sampling. All other animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test article-related effects upon mean body weights were noted in either sex at any dose levels. The mean body weight gain values of the test article-treated males and females were similar to or exceeded those of the controls.
Food efficiency:
no effects observed
Description (incidence and severity):
The mean daily food consumption of all groups was similar.
Haematological findings:
no effects observed
Description (incidence and severity):
No test article-related differences to the control values were noted in males or females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test article-related differences to the control values were noted at any dose level.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test article-related differences to the control values were noted at any dose level.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test article-related differences to the control animals were noted at any dose level.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test article-related changes were noted in the absolute or relative organ weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings after 4 weeks' treatment or two weeks' recovery.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The administration of the test substance, by gavage, to Wistar rats, at dosages of 50, 200 or 1000 mg/kg/day for 28 days produced no treatment-related findings.

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 effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The highest dose applied, 1000 mg/kg bw/day, was determined to be the NOAEL and the NOEL as no effects were observed.