P-1401

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Test animals

Species:

rat

Strain:

other: HanRcc:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Males: 178-194 g at acclimatization (mean 185 g), Females: 134-147 g at acclimatization (mean 139 g).
- Housing: In groups of five in Makrolon type-4 cages with wirenmesh tops and standardized softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 (batch no. 67/06) rat maintenance diet (Provimi Kliba AG, CH-4303 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: 7 days. Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily. 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. Homogeneity of the test item in the vehicle was 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:

Concentration, homogeneity and stability (after 2 hours) of the dose formulations were determined in samples taken after experimental start. 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.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based upon the results of a 5-day doserange-finding study in which the test item was administered by gavage to 2 rats per group and sex.

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.

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 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 the pretest and treatment periods and before necropsy.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum)
- How many animals: All animals
- Parameters: 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) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Heinz bodies (to be completely assessed only if changes in methemoglobin are noted), 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
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum)
- How many animals: All animals
- Parameters: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein total, Albumin, Globulin, Albumin/Globulin ratio

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations: Week 4
- Dose groups that were examined: All animals
- Battery of functions tested:
Grip Strength
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.
Locomotor Activity
Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were 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 10-minute intervals as well as the total activity of the measuring period.

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, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl coagulating gland), Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions.

ORGAN WEIGHTS: Yes. Brain, Thymus, Spleen, Ovaries, Heart, Kidneys, Testes, Liver, Adrenals, Epididymides

HISTOPATHOLOGY: Yes. Slides of all organs and tissues listed that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (kidneys and lungs) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory investigations, organ weights and ratios:
• 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.
• The Steel-test (many-one rank test) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test were applied to the macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related findings were noted during daily observations at any dose level tested.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were noted in the mean body weights and body weight gains of animals treated with 800 mg/kg body weight/day. Animals treated with 800 mg/kg body weight/day showed reduced mean body weights and body weight gains compared to controls with statistical significance in females (p<0.01). These dose-related changes were accompanied by changes in food consumption and were considered to be test item-related.
The mean body weights of the males and females treated with 200 mg/kg/day and 50 mg/kg/day were considered to be unaffected.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
At 800 mg/kg body weight/day, reduced absolute food consumption was noted in males and females and reduced relative food consumption were seen in females only, when compared to controls. Males treated with 200 mg/kg body weight/day showed reduced absolute food consumption towards the end of the treatment phase compared to controls. The mean food consumption of the animals treated with 50 mg/kg/day was unaffected.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were noted in the hematology parameters of either sex at any dose level tested.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings indicative of changes in liver metabolism and kidney parameters were noted in animals treated with 800 and 200 mg/kg body weight/day. At 800 mg/kg/day, these changes included increased creatinine levels were noted in males and some females; increased aspartate aminotransferase activity in males and females; increased cholesterol in males; decreased triglycerides in males; decreased potassium in males and females, and decreased albumin levels in females. At 200 mg/kg/day, these changes included elevated creatinine in males and females, elevated cholesterol in males and elevated phospholipids in males. No changes of toxicological relevance were noted at 50 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related findings were noted during the functional observational battery (week 4) at any dose level tested.
Grip Strength
No test item-related changes in the mean grip strength were noted at any dose level tested.
Locomotor Activity
No test item-related changes in the mean locomotor activity were noted at any dose level tested.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative kidney weights were elevated in animals treated with 800 and 200 mg/kg body weight/day compared to controls and considered to be test item-related. Absolute and relative thymus weights were decreased in animals treated with 800 and 200 mg/kg body weight/day and to a lesser degree in animals treated with 50 mg/kg body weight/day. These findings were considered to be test item-related but were not accompanied by relevant microscopical changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Tan discoloration was recorded in the kidneys of one male treated with 200 mg/kg/day and in all animals treated with 800 mg/kg/day. This finding was deemed to be test item related and correlated with histological kidney changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, changes could be found in the kidneys of animals treated with 200 mg/kg/day and 800 mg/kg/day. These changes consisted of a combination of vacuoles in tubular epithelial cells (containing fine granular material), tubular cell necroses, tubular basophilia including tubular regeneration, interstitial nephritis and tubular casts.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The no-observed-adverse-effect-level (NOAEL) was considered to be 800 mg/kg/day.

Executive summary:

Oral administration of the test item to Wistar rats at doses of 50, 200 and 800 mg/kg/day, for 28 days resulted in no deaths, no clinical signs during daily observations, no clinical signs during weekly observations (weeks 1-3), no clinical signs during the functional observational battery (including no changes in mean grip strength or locomotor activity). Test item-related findings were generally restricted to reduced mean daily food consumption in both sexes at 800 mg/kg/day and in males at 200 mg/kg/day, reduced mean body weights and mean body weight gain in males and females treated with 800 mg/kg/day, changes in clinical biochemistry parameters that were generally indicative of alterations in liver metabolism and kidney function at 800 mg/kg/day and 200 mg/kg/day, changes in absolute and relative kidney weights at 800 mg/kg/day and 200 mg/kg/day, as well as macroscopical and microscopical findings in the kidneys of rats treated with 800 mg/kg/day and 200 mg/kg/day. Reduced mean absolute and relative thymus weights noted in rats treated with 800 mg/kg/day 200 mg/kg/day and to a lesser extent in animals treated with 50 mg/kg/day.

Chronic progressive nephropathy (CPN) is a rodent-specific, age-related renal disease, particularly of male rats, characterized by a spectrum of distinct histological changes which may begin early in the animal's life and progress to end-stage renal disease in certain rat strains. CPN is generally regarded as a degenerative to atrophic disease with compensatory regenerative hyperplasia. The proliferative nature of CPN often becomes problematic in advanced to end-stage renal disease. CPN is a rodent specific disease with no apparent similar human kidney disease condition.

Therefore, the no-observed-adverse-effect-level (NOAEL) was considered to be 800 mg/kg/day.