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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:
2007-09-28 to 2008-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (OECD 407 adopted 1995), GLP compliant. Acceptable deviations fromOECD Guideline 407 adopted 2008: less organs weighed and mircoscopically examined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 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
Deviations:
no
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: RCC Ltd Laboratory Animal Services; Wölferstrasse 4; 4414 Füllinsdorf / Switzerland
- Total Number of Animals Used: 30 males and 30 females
- Age (at Delivery): 7 weeks
- Body Weight Range: Males: 158.3 to 170.2 g (mean 164.0 g) ; Females: 128.1 to 145.0 g (mean 135.1 g)
- Acclimation: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding
- Diet: Pelleted standard Kliba Nafag 3433 (batch no. 40/07) rat maintenance diet was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): microgranulated feed; an appropriate amount of water was added to aid pelleting; the pellets were dried with air for approximately 48 hours before storage.
- Storage temperature of food: room temperature (20 ± 5 °C)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After experimental start, samples of the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of homogeneity, concentration and stability. Samples of each concentration were taken during weeks 2 to 4 after each adjustment of feed preparations (based on previously measured body weight gain and feed consumption) to confirm homogeneity and concentration. Analysis done by High Performance Liquid Chromatographic demonstrated a stability of 12 days and confirmed homogeneity and concentrations.
Duration of treatment / exposure:
28 days
Frequency of treatment:
continuous
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 52, 215, 1063 (males) and 0, 52, 207, 1020 (females) mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
Main test: 5 males / 5 females per dose;
Reccovery: additional 5 animals/sex at 0 and at 1000 mg/kg bw/day
Control animals:
yes, plain diet
Details on study design:
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Viability/Mortility: twice daily
- General clinical observations: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly during week 1, once weekly during week 2 to 4

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: Twice weekly during week 1, once weekly during week 2 to 4
- Food consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes, 18 hours fasting period
- How many animals: all
- Parameters examined: Erythrocyte count
Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium,high fluorescence), Leukocyte count, total, Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Animals fasted: Yes, 18 hours fasting period
- How many animals: all
- Parameters examined: Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-ransferase, Creatine kinase, Sodium Potassium, Chloride, Calcium, Phosphorus, Protein, total, Albumin, Globulin, Albumin/Globulin ratio, Prothrombin time (= Thromoplastin time), Activated partial Thromoplastin time, Methemoglobin

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 and 6 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (urine collected during 18 hours fasting period)
- Parameters examined: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance, pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all
- Battery of functions tested: modified Irwin screen incl. sensory activity / grip strength / motor activity
Sacrifice and pathology:
- Sacrifice:
After 4 Weeks: Main test
After 6 Weeks: Recovery

GROSS PATHOLOGY: Yes
- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.
- Organ weights dertemined: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Thyroid / parathyroid, Uterus

HISTOPATHOLOGY: Yes
- Tissues examined: Adrenal glands; Bone marrow (femur); Brain - including section of medulla/pons, cerebral and cerebellar cortex; Cecum; Colon; Duodenum; Epididymides (fixed in Bouin's solution); Heart including auricles; Ileum, with Peyer's patches; Jejunum with Peyer's patches; Kidneys; Liver; Lungs, filled w/formalin at necropsy; Lymph nodes - mesenteric and mandibular; Ovaries; Prostate gland incl. coagulating glands; 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, filled w/formalin at necropsy; Uterus; Vagina; Gross lesions;
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• 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 Dunnetttest when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy. No test item-related clinical signs were noted.

BODY WEIGHT AND WEIGHT GAIN
No test item-related effects on the absolute or relative body weights were noted at any dose level tested.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test item-related effects on the absolute and relative food consumption was noted. The mean of means of the test item intake over treatment period was close to all target doses. It ranged from 103.8% to 107.7% in males and 102.0% to 103.4% in females.

HAEMATOLOGY
No test item-related changes were noted in the hematology parameters of either sex after treatment and revovery period at any dose level tested.

CLINICAL CHEMISTRY
Test item-related adaptive changes were noted in males and females treated with 1000 mg/kg body weight/day and in females treated with 200 mg/kg body weight/day.
In animals treated with 1000 mg/kg body weight/day elevated sodium, albumin and protein was noted and additionally elevated cholesterol, calcium and decreased phoshorus in females, only. Females treated with 200 mg/kg body weight/day showed elevated sodium, only.
No changes were noted in enzymes indicating liver or kidney damage.
No test item-related changes were noted after recovery period.

URINALYSIS
No test item-related changes were noted.

NEUROBEHAVIOUR
- Functional observation battery and grip strength: No test item-related findings were noted.
- Locomotor activity: Initial elevation of the locomotor activity was noted at the first two timepoints of measurement in animals treated with 1000 mg/kg body weight/day. Nevertheless, during the detailed behavioral observations, no clinical signs were noted which would have been indicative of possible neurotoxicity. Therefore this finding was considered to be non-adverse.

ORGAN WEIGHTS
No test item-related changes were noted in the mean absolute or relative organ weights at the end of treatment or recovery period.

GROSS PATHOLOGY / HISTOPATHOLOGY
No test item-related macroscopic or microscopic findings were noted at the end of treatment or recovery period at any dose level tested.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased albumin, protein, cholesterol and calcium levels and decreased phosphorous levels at 1000 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects seen in lower doses are considered to be adaptive processes or were not noted after recovery period anymore, and therefore non-adverse

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dietary administration of the test item at target dose levels of 50, 200 and 1000 mg/kg body weight/day had no effects on survival rate, clinical signs, grip strength, food consumption, body weights, hematology, urinalysis parameters, organ weights and macroscopic or microscopic changes in pathology.

In clinical biochemistry, slight increased sodium levels in both sexes treated with 1000 mg/kg body weight/day and in females treated with 200 mg/kg body weight/day were noted but not accompanied by changes in hematocrit, urinalysis parameters such as volume or density or changes of the adrenal gland in histopathology. The changes stayed within ranges of historical reference data and were not noted after recovery period. Increased sodium levels were considered to be test item-related but non-adverse.

In animals treated with 1000 mg/kg body weight/day increased levels of albumin and protein were above ranges of historical reference data in females. These changes in albumin and protein levels were not accompanied by increased hematocrit indicating dehydration and therefore considered to be an adaptive process of the liver. In absence of microscopic findings, changes in liver specific enzymes and findings after recovery period, the findings of elevated albumin and protein were considered to be test item-related but non-adverse. An interference of a possible coloring effect of the test item with the colorimetric method of measuring albumin and protein in plasma could not be outruled.

Elevated cholesterol, calcium and decreased phosphorus in high dose females were not noted after recovery period anymore and also considered to be test item-related but non-adverse.

Initially elevated locomotor activity in males and females treated with 1000 mg/kg body weight/day was not accompanied by any test item-related clinical signs and was therefore considered to be non-adverse.

Based on the results of this study, 1000 mg/kg/day of the test item was established as the no-observed-adverse-effect-level (NOAEL) and 50 mg/kg body weight/day as the no-observed-effect-level (NOEL).

Applicant's summary and conclusion