Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, OECD guideline conform, well documented, read across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: HanBrhWIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Fuellinsdorf / Switzerland
- Age at study initiation: 7 weeks
- Weight at acclimatization: Males: 129.6 - 161.0 grams (mean 139.8 grams), Females: 109.7- 130.7 grams (mean 119.1 grams)
- Housing: groups of five animals
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 (batch nos. 119/01 and 05/02) rat maintenance diet (Provimi Kliba AG, Kaiseraugst/ Switzerland) was available ad libitum.
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Lot/batch no. (if required): 433337/1
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test item/vehicle mixtures were prepared and about 2 g (weighed to the third decimal place) of each mixture was weighed into a 100-ml volumetric flask by RCC Ltd, Toxicology Division, Itingen. These samples for analysis of concentration and homogeneity were delivered to the analytical laboratory where they were stored deep-frozen (about -20 °C) until analysis. Storage stability samples were collected after storage (2 hours, 7 days) and then delivered to the analytical laboratory where they were stored deep-frozen until analysis.
The delivered samples were dissolved in about 70 ml of acetonitrile/tetrahydrofuran (1-t-l v/v) using an ultrasonic bath. Then, the 100-ml volumetric flasks were filled to the mark with acetonitrile/tetrahydrofuran (1-1-1 v/v). Depending on the dose group, the latter sample solutions were further diluted with acetonitrile/tetrahydrofuran (l-i-l v/v) to yield concentrations within the calibration range. Finally, a defined aliquot was quantified by HPLC.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200, 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 animals; additional five animals per sex were available in groups 1 (controls) and 4 (high dose) for the assessment of reversibility of effects.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon the results of a non-GLP 5-day doserange-finding study (RCC Study Number 842521) in which the test substance was administered by gavage to 2 rats per group and sex.
Positive control:
no

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period
- Anaesthetic used for blood collection: Yes, light isoflurane
- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: 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: At the end of the dosing and the treatment-free recovery period
- Animals fasted: Yes, in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- 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, Albumin, Protein total, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the dosing and the treatment-free recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for approximately 18 hours before sampling
- Parameters examined: Volume (18-hour), Specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Nitrite, Urobilinogen, Urine sediment, Red blood cells, White blood cells, Crystals (Triple phosphate)

OTHER:
GRIP STRENGTH: Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AGF 25N).

LOCOMOTOR ACTIVITY: Locomotor (decreased or increased) activity was measured quantitatively with Activity Monitor AM 1052 system (Benwick Electronic Equipment Design Manufacture, England). Animals were randomized and monitored during the fourth treatment week for a 60-minute
period and the total activity of this time period was recorded.
Low beams count was reported in 15-minute intervals as well as the total activity of the measuring period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as clinical laboratory data :
• 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 grip strength and locomotor activity.
• Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
transient, slightly decreased locomotor activity in high dose group
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No test item-related clinical signs of toxicological relevance were noted during daily or weekly observations (weeks 1-3). All animals survived until scheduled necropsy.

FOOD CONSUMPTION AND BODY WEIGHT : No test item-related changes in food consumption or body weight were noted at any dose
level tested.

HAEMATOLOGY: No test item-related changes of toxicological significance were noted after treatment or recovery in the hematology parameters at any dose level.

CLINICAL CHEMISTRY: The clinical biochemistry parameters of the test item-treated rats were similar to those of the controls after the treatment and recovery periods.

URINALYSIS: The urinalysis parameters of the test item-treated rats were unaffected after the treatment
and recovery periods

ORGAN WEIGHTS: No test item-related differences in the absolute or relative organ weights were noted at any dose level after four weeks' treatment or two weeks' recovery.

GROSS PATHOLOGY: No test item-related macroscopic or microscopic changes were noted at any dose level after the treatment or recovery periods.

OTHER FINDINGS:
FUNCTIONAL OBSERVATIONAL BATTERY: No test item-related clinical signs of toxicological relevance were noted during functional observations (week 4).

GRIP STRENGTH: No test item-related differences in fore- or hindlimb grip strength values were noted at any dose level tested.

LOCOMOTOR ACTIVITY: Slight reductions in mean locomotor activity were noted in both sexes treated with 1000 mg/kg/day. These differences were transient, but contributed to slightly lower total locomotor activity on both sexes and was considered to be a minor effect related to the test item.
The locomotor activity values of the rats treated with 50 mg/kg/day or 200 mg/kg/day compared favorably with those of the controls.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed.
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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Repeated dose application of the test item did not induce mortalities, signs of toxicity or any abnormalities. Animals of the high dose group showed slightly lower locomotor activity compared to lower dose groups and control animals. The decrease in activity was present in the first 15 min only, thereafter the high dose animals behave similar to control animals. A dose-effect relationship was not observed; a confirmatory test was not conducted. Thus, the change in locomotor activity is regarded as an unspecific high dose effect rather than a treatment related symptom.