Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-04 to 2005-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study (OECD 407 adopted 1996) with acceptable deviations from OECD guideline 407 adopted 2008: less organs were weighed and microscopically examined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
September 30, 1996
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
July 27, 1995
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals : Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Description: Red solid
- Storage conditions: at room temperature (20 +/- 5 °C) in the original container, away from direct sunlight

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd.; Laboratory Animal Services; CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 6 weeks;
- Weight at study initiation: Males: 140.0 - 163.3 grams (mean 153.9 grams; Females: 117.0-137.8 grams (mean 124.1 grams);
- Fasting period before study: No
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland)
- Diet: Pelleted standard Provimi Kliba 3433 (batch no. 53/04 and 63/04) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) ad libitum;
- Water: Community tap-water from Itingen was available ad libitum in water bottles
- Acclimation period: 7 days;

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C;
- Humidity: relative humidity range: 30-70 %;
- Air changes: 10-15 air changes per hour;
- Photoperiod: 12-hour fluorescent light/12-hour dark cycle with music during the light period.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
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 (weight/volume). The mixtures were prepared using a magnetic stirrer and stored at room temperature (15-25°C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: solubilty of the test item;
- Amount of vehicle (if gavage): 5 mL/ kg body weight;

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken on the day of animal delivery. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed according to a HPLC method.
The results demontrated that the nominal concentrations wer achieved, that the test item was homogeneously distributed in the vehicle and stable in the vehicle under storage conditions for two hours and for seven days.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg body weight; Group 2: 50 mg/kg body weight; Group 3: 200 mg/kg body weight; Group 4: 1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose in treatment groups; 5 animals per sex per dose in the recovery groups (0 and 1000 mg/kg bw)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of a non-GLP 5-day dose range-finding study
- Post-exposure recovery period in satellite groups: 14 days

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; as well as once daily on days 4-28 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.

FOOD CONSUMPTION AND COMPOUND INTAKE: The food consumption was recorded once during the pretest period and weekly thereafter.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks and after 6 weeks (recovery)
- Anaesthetic used for blood collection: yes (isoflurane)
- Animals fasted: Yes
- How many animals: All
- Parameters checked/examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) countReticulocyte count, Reticulocyte maturity index, Methemoglobin, Total leukocyte count, Differential leukocyte count, Coagulation:, Thromboplastin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks and after 6 weeks (recovery)
- Anaesthetic used for blood collection: yes (isoflurane)
- Animals fasted: Yes
- How many animals: All
- Parameters checked/examined: Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferas, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, total, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: during the 18-hour fasting period
- Metabolismus cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked/were examined: Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4;
- Dose groups that were examined: all dose groups;
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated):
Adrenal glands (weighed), Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 levels)(weighed), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution)(weighed), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart(weighed), Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys(weighed), Larynx, Lacrimal gland (exorbital), Liver(weighed), Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries(weighed), Pancrea, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen(weighed), Stomach, Testes (fixed in Bouin's solution)(weighed), Thymus(weighed), Thyroid (incl. parathyroid gland, if possible), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina,Gross lesions

The following organ weights were recorded on the scheduled dates of necropsy:
Brain, Heart, Liver, Thymus, Kidneys, Adrenals, Spleen, Testes, Epididymides, Ovaries


HISTOPATHOLOGY: Yes
- Slides of all organs and tissues listed were collected at scheduled sacrifice from the animals of control and high-dose groups and were examined by a pathologist: Adrenal glands, Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Heart, Ileum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Ovaries, Prostate gland, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland, if possible), Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions
Other examinations:
NA
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were 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.
• Fisher's exact-test were applied to the macroscopic findings.

The following statistical methods were used for statistical analysis of clinical laboratory data:
• Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p<0.05), a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% level and by Dunn's test in the case of a significant Kruskal-Wallis test (p<0.05).

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until their respective scheduled necropsy.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights and the mean body weight gain of the test item-treated rats compared favorably with those of the respective controls.

FOOD CONSUMPTION
The mean daily food consumption values of the test item-treated rats were unaffected during the treatment and recovery periods when compared with the control rats.

HAEMATOLOGY
No test item-related differences of toxicological relevance were noted in the hematology parameters after treatment or recovery.

CLINICAL CHEMISTRY
No test item-related differences of toxicological relevance were noted in the clinical biochemistry parameters after treatment or recovery.

URINALYSIS
No test item-related differences of toxicological relevance were noted in the urine parameters after treatment or recovery.

NEUROBEHAVIOUR
No test item-related findings of toxicological relevance were noted during functional observational battery (week 4) at any dose level.

ORGAN WEIGHTS
No test item-related differences of toxicological relevance were noted in the mean absolute or relative organ weights at any dose level tested.
All differences in the mean absolute or relative organ weights noted after recovery were considered to be incidental and unrelated to the test item administration.

MACROSCOPIC / MICROSCOPIC FINDINGS
At necropsy, no test item-related macroscopic findings were recorded.
Microscopically, no test item-related histopathological findings were recorded.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion