Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-06-12 to 2006-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
HT001492 IRA161
- Expiration date of the lot/batch:
2010-06-20
- Purity test date:
no data


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature (15-25°C) away from direct sunlight
- Solubility and stability of the test substance in the solvent/vehicle:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Analyses were performed by the Principal Investigator of the analytical phase according to a HPLC analytical method supplied by the Sponsor and previously adapted at RCC Ltd (RCC Study Numbers A46618 and A71447).

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
6 weeks
- Weight at study initiation:
Males: 136.8 - 154.9 grams (mean 146.1 grams); Females: 118.0 - 132.6 grams (mean 125.7 grams)
- Fasting period before study:
No.
- Housing:
In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding.
- Diet (e.g. ad libitum):
Pelleted standard Provimi Kliba 3433.
- Water (e.g. ad libitum):
Community tap-water from Itingen was available ad libitum in water bottles
- Acclimation period:
For 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 °C
- Humidity (%):
30-70%
- Air changes (per hr):
10-15 air changes per hour
- Photoperiod (hrs dark / hrs light):
12-hour fluorescent light/12-hour dark cycle
- Other: music during the light period

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Method: Oral gavage
Frequency of administration: daily
Vehicle:
polyethylene glycol
Remarks:
; PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared weekly. T001492 was weighed into a glass beaker on a tared Mettler balance and the vehicle added and homogenized. 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 (if other than water):
No information available.
- Concentration in vehicle:
0 mg/mL (group 1); 2 mg/mL (group 2); 6 mg/mL (group 3); 20 mg/ mL (group 4)
- Amount of vehicle (if gavage):
5 ml/kg body weight
- Purity: No data
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 after experimental start. Analyses were performed by the Principal Investigator of the analytical phase), according to a HPLC analytical method supplied by the Sponsor and previously adapted at RCC Ltd (RCC Study Numbers A46618 and A71447). Details of the analytical method are documented in the raw data generated by the Principal Investigator (and/or his/her staff) and the Principal Investigator provided an analytical phase report. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The dose formulations were delivered under ambient conditions. Samples of dose formulations were not discarded without the written authorization of the study director.
Duration of treatment / exposure:
28 days of treatment with 14 days of recovery
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control group)
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
Groups 1 and 4: 10 males and 10 females
Groups 2 and 3: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based upon the results of a non-GLP 5-day dose-range-finding study (RCC Study Number A71447) in which T001492 was administered by gavage to 2 rats per group and sex.
- Rationale for animal assignment (if not random): Randomized
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; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).
- Mortality/viability: Twice daily.

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 AND COMPOUND INTAKE (if feeding study):
- 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
- Blood sampling: After 4 weeks: 17 July 2006. After 6 weeks: 31 July 2006.
- Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane 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.
- 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, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Heinz bodies (not determined because no changes in methemoglobin were noted), Total leukocyte count, Differential leukocyte count, Coagulation: Thromboplastin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Blood sampling: After 4 weeks: 17 July 2006. After 6 weeks: 31 July 2006.
- Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane 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.
- Parameters checked: 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.


URINALYSIS: Yes
- Time schedule for collection of urine:
After 4 weeks: 17 July 2006. After 6 weeks: 31 July 2006. Urine was collected during the 18-hour fasting period into a specimen vial.
- Parameters checked: Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.

FUNCTIONAL OBSERVATIONS:
- During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
- Parameters examined: Locomotor (decreased or increased) activity, Forelimb and hind limb grip strength
Sacrifice and pathology:
SACRIFICE:
- After 4 weeks (on 17 July 2006) and after 6 weeks (on 31 July 2006).
- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: Yes
- Samples of the tissues and organs below were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (unless otherwise indicated).
- Tissues and organs collected: 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, midthoraclc, 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.


HISTOPATHOLOGY: Yes
- All organ and tissue samples, as defined under Histopathology (below), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.
- Histopathology: Slides of all organs and tissues listed in boldface type in the study report that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. Following preliminary examination, the livers from the animals of the mid-dose groups (10 and 30 mg/kg/day) were examined because of test item-related findings noted in the animals treated with 100 mg/kg/day.

Other examinations:
ORGAN WEIGHTS:
- The following organ weights were recorded on the scheduled dates of necropsy: Brain, Heart, Liver, Thymus, Kidneys, Adrenals, Spleen, Testes, Epididymides, Ovaries.
- The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
- The determination of the terminal body weight was performed immediately prior to necropsy.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory data, organ weights and ratios:
• 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.
• Fisher's exact-test was applied to the macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were noted during the detailed clinical observations performed during weeks 1- 3 of treatment.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences between control animals and test item treated animals in body weight or body weight gain were noted.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effect of the test item on food consumption was noted. Differences in absolute and relative food consumption between groups were within the range of normal biological variation for rats of this strain and age.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related changes were seen in hematological parameters.

In males and females treated with 100 mg/kg/day, statistically significant increases in hemoglobin concentration (p<0.01) and hematocrit (p<0.05) were noted after 4 weeks of treatment. These increases were within the normal range for rats of this strain and age. No differences in hematology parameters were seen after recovery.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related changes were seen in clinical biochemistry parameters.

In males and females treated with 100 mg/kg/day, a minor decrease in total bilirubin (statistically significant in females, p<0.05) and a minor statistically significant increase in triglycerides (p<0.01) was noted. Additionally, a minor dose related increase in potassium (statistically significant, p<0.05) and a dose related increase in phosphorus was found in females treated with 100 mg/kg/day. After recovery, a minor increase in triglycerides and a minor decrease in phosphorus (p<0.05) were noted in males treated with 100 mg/kg/day. In females treated with 100 mg/kg/day, a minor decrease of total bilirubin and a minor increase in aspartate aminotransferase and alkaline phosphatase (each statistically significant, p<0.05) were found. However, these and all other changes seen were within the normal range of biological variation for rats of this strain and age.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related changes were seen in urinalysis.
In urinalysis after 4 weeks, increases in relative density (p<0.05), protein (p<0.05), bilirubin (not significant), and leukocytes (p<0.05) were noted in females treated with 100 mg/kg/day. The increase in leukocytes was not dose related and mainly due to very high leukocytes in one single animal. All other changes were within the normal range of biological variation for rats of this strain and age. No changes in urinalysis parameters were seen after recovery.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item related differences in the functional observational battery were noted at week 4 of treatment.

Grip Strength: No test item-related differences were noted in the mean grip strength when compared with the controls. Hindlimb grip strength in females treated with 30 or 100 mg/kg/day was significantly higher (p<0.05) than in controls. As this finding was not dose related and the control grip strength was low, this is considered to be incidental.

Locomotor Activity: No test item-related differences were noted in the mean locomotor activity when compared with the controls. Significantly decreased locomotor activity was seen from 40-50 min. in males treated with 30 mg/kg/day (p<0.05) and females treated with 100 mg/kg/day (p<0.05). A statistically significant increase was seen from 0-10 min. in males treated with 100 mg/kg/day (p<0.01) and from 20-30 min. in females treated with 10 mg/kg/day (p<0.05). These observations were neither dose related nor consistent across sexes, and are therefore considered to be incidental.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 4 Weeks: A slight increase in absolute and relative liver weights statistically significant in organ to body weight ratio (p<0.05) was found in males and females treated with 100 mg/kg/day.

After 6 Weeks: A very slight increase in absolute (p<0.05 in females) and relative (organ to brain weight ratio) liver weights of males and females treated with 100 mg/kg/day persisted but the difference to controls was less than at week 4.
A statistically significant increase in absolute (p<0.0.1) and relative (p<0.05 and p<0.01, respectively) adrenal weights and a statistically significant decrease in absolute and relative testes weights (p«0.05 each) compared to controls were noted in males treated with 100 mg/kg/day. Thymus weights in males and females treated with 100 mg/kg/day tended to be higher than in controls but lower than in the corresponding animals after 4 weeks.

The slight statistically significant increase in the liver-to-body weight ratio in males and females treated with 100 mg/kg/day was attributed to metabolic adaptation and considered not to be adverse.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 4 weeks, foci in the thymus were noted in one male of the control group and one female treated with 30 mg/kg/day. Discoloration of the thymus and one mandibular lymph node was found in one male treated with 100 mg/kg/day. Unilateral renal pelvis dilation was noted in another male treated with 100 mg/kg/day.

After 6 weeks, foci in the thymus were noted in one male of the control group and discoloration of the thymus was found in one male treated with 100 mg/kg/day. Unilateral renal pelvis dilation and a watery cyst in the ovaries were found in one female treated with 100 mg/kg/day.

These findings are within the range of normal background lesions, which may be commonly recorded in animals of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular hypertrophy of hepatocytes was found in 3/5 males treated with 100 mg/kg/day at minimal to slight severity, in 2/5 males treated with 30 mg/kg/day at slight severity, and in one female treated with 100 mg/kg/day at slight severity. After the recovery period, the livers of test item treated animals were comparable to those of controls. All other microscopic observations recorded in this study were within the normal range of physiological changes and background alterations that may be seen in untreated animals of this age and strain. Hepatocellular hypertrophy is a common adaptive response and in the absence of other signs of hepatotoxicity considered to be non-adverse.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
- Analysis of dose preparations:
The application formulations investigated during the study were found to comprise T001492 in the range from 92.9% to 122.7%. except for one sample result the required content limit of +/-20% with reference to the normal concentration was not exceeded. The homogenous distribution of T001492 in the preparations was approved because single results found deviated not more than 12% from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept 2 hours and 7 days under storage conditions due to recoveries which meet the variation limit of 10% from time- zero (homogeneity mean).

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Treatment-related findings were generally restricted a slight increase in the liver-to-body weight ratio in males and females treated with 100 mg/kg/day attributed to metabolic adaptation. Both findings were considered not to be adverse. In accordance with increased liver weights, hepatocellular hypertrophy was found especially in males treated with 30 or 100 mg/kg/day but also in one female treated with 100 mg/kg/day. Hepatocellular hypertrophy is a common adaptive response and in the absence of other signs of hepatotoxicity considered to be non-adverse.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral administration of T001492 to Wistar rats at doses of 10, 30 and 100 mg/kg/day, for 28 days resulted in no unscheduled deaths. Treatment-related findings were generally restricted to salivation in three males treated with 100 mg/kg/day and a slight increase in the liver-to-body weight ratio in males and females treated with 100 mg/kg/day attributed to metabolic adaptation. Both findings were considered not to be adverse. In accordance with increased liver weights, hepatocellular hypertrophy was found especially in males treated with 30 or 100 mg/kg/day, but also in one female treated with 100 mg/kg/day. Hepatocellular hypertrophy is a common adaptive response and in the absence of other signs of hepatotoxicity considered to be non-adverse.

Based on the results of this study, 10 mg/kg body weight/day of T001492 was established as the no-observed-effect-level (NOEL) based on the non-adverse, adaptive liver responses, and 100 mg/kg body weight/day of T001492 as the no-observed-adverse-effect-level (NOAEL).

The substance is not classified as a repeated dose toxicoant (STOT RE) according to the CLP Regulation.