Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-08-24 to 2001-10-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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
Remarks:
in compliance with the Swiss Ordinance relating to GLP, adopted February 2nd, 2000 [RS 813.016.5], based on the OECD principles of GLP, adopted November 26th, 1997 [C(97) 186/Final]
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): T002113
- Substance type: powder
- Physical state: solid
- Analytical purity: 92%
- Purity test date: no data
- Lot/batch No.: RT002113G3A231
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable under storage conditions
- Retest date: 01 December 2001
- Storage condition of test material: at room temperature (17-23 °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: HanBrl:WIST (SPF), RCC Ltd, Biotechnology & Animal Breeding Division, CH-4414 Füllinsdorf, Switzerland
- Age at delivery: 6 weeks
- Weight at acclimatization: 134-154 grams for males (mean 144 grams), 109-129 grams for females (mean 118 grams)
- Fasting period before study: no data
- Housing: in groups of five in Makrolontype-4 cages with wire mesh tops and satndardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland)
- Diet (e.g. ad libitum): ad libitum, pelleted standard Provimi Kliba 3433 (batch no. 73/01) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland). The feed batch was analyzed for contaminants.
- Water (e.g. ad libitum): ad libitum, community tap water from Ittingen in water bottles. Bacteriological assay, chemical and contaminant analyses of representative samples performed
- None of the contaminants analyzed in the water and diet is considered to have been present at a concentration which would have affected the validity of
the results
- Acclimation period: 7 days, 31 August 2001 to 06 September 2001, under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (light period between 06.00 and 18.00), music during light period

IN-LIFE DATES: From: 31 August 2001 To: 05 October 2001

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: bi-distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared weekly. T002113 was weighed into a glass beaker on a tared Mettler balance and the vehicle added (weightvolume). 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.

DIET PREPARATION: no data


VEHICLE
- Amount of vehicle (if gavage): 10 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 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 by RCC Ltd (Environmental Chemistry & Pharmanalytics
Division) according to a HPLC method supplied by the Sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
450 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for test system: recognized by the international guidelines as the recommended test system
- Dose selection rationale: Based upon the results of a non-GLP 5-day doserange-finding study (RCC Study Number 825153) in which T002113 was administered by gavage to 2 rats per group and sex. Animals showed effects on food consumption and body weight at 600 mg/kg/day and 1000 mg/kg/day and clinical signs of toxicity at 1000 mg/kg/day.
- rationale for administration route: accidental oral ingestion is a possible route of human exposure

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: These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.
- The animals were observed in their home cages, outside their home cages in a standard arena and in the hand.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest and treatment 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):
- Food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettles balance connected to the RCC computer.
- Data calculation: food consumption calculated per rat and per food consumption interval, expresses the average food consumed per animal and per day over the food consumption interval. Relative food consumption caclulated in grams of food per kg body weight and day

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks, early in the working day to reduce biological variation caused by circadian rhythms, drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- Anaesthetic used for blood collection: Yes (light isoflurane)
- 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 examined, included 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) and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks, early in the working day to reduce biological variation caused by circadian rhythms, drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube
- 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
- Anaesthetic used for blood collection: Yes (light isoflurane)
- Parameters examined, included glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl transferase, calcium, phosphorus, sodium, potassium, chloride, albumin, total protein, globulin and the albumin/globulin ratio.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength (forelimb and hind limb grip strength), locomotor activity, appearance (salivation, hunched posture, neck alopecia, kink on tail tip), motor (ataxia, tremor/twitching, prostration, circling, spasm), behavior (hyperactivity, somnolence, increased exploration, reduced grooming, vocalisation), respiration (dyspnea, tachypnea, bradypnea), reflexes (blink, pinna, iridic light reflex, push-off (hind leg), pain response, startle/hearing), miscellaneous (lacrimation, limbs cyanotic, mydriasis, miosis, exophthalmos, reduced muscle tone, beddinq dark oranqe)

OTHER: no data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were weighed and necropsied after 4 weeks. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination. 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 (cerebrum, cerebellum, pons), 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, 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 WEIGHT:
- 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.

HISTOPATHOLOGY: Yes
- Slides of all the following organs and tissues which were collected at scheduled sacrifice from the animals of control and high-dose groups were
examined by the study pathologist: adrenal glands, bone marrow (femur), brain (cerebrum, cerebellum, brain stem), cecum, colon, duodenum, epididymides, heart, ileum (with Peyer's patches), jejunum (with Peyer's patches), kidneys, liver, lungs, lymph nodes (madibular, mesenteric), ovaries, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, testes, thymus, thyroid (incl. parathyroid) gland, trachea, urinary bladder, uterus, vagina, and all gross lesions
- Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group.
- Because test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (livers and thyroids) 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, 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.
- Fisher's exact-test was applied to the macroscopic findings.
- Student's t-test was applied to grip strength and locomotor activity

References:
- C W Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R G Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R A Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Dark orange bedding, noted from day 9 onwards in the males and females treated with 450 mg/kg/day, was considered to be a test item-related effect possibly caused by an unspecified urinary metabolite. All remaining findings (salivation, kinked tail, localized alopecia) were considered to be incidental findings.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female (no. 38) treated with 450 mg/kg/day died on treatment day 11. All other animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower body weights and reduced body weight gain was noted in the males treated with 450 mg/kg/day. This finding was considered to be test-item related. The body weights of the remaining animals were unaffected.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A transient reduction of food consumption was noted in the females treated with the test item at 450 mg/kg/day (week 1 only). This finding was considered to be test-item related. All other animals were 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 changes were noted in the haematology parameters when compared with the controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No test-item related changes were noted in the clinical biochemistry parameters of males when compared with the controls.
Test item-related changes in the clinical biochemistry parameters of females treated with 450 mg/kg/day included increased levels of total cholesterol and phospholipids, and higher activities of alanine aminotransferase and gamma glutamyltransferase, and were considered to be evidence of changes in liver metabolism. All other animals were unaffected.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test-item related clinical signs of toxicological relevance were noted at any dose level.

Grip strength
The mean fore-and hindlimb grip strength of the test item-treated rats was unaffected.

Locomotor activity:
No test-item related differences in locomotor activity were noted at any dose level.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes included increased liver weights in males and females treated with the 450 mg/kg/day. The trend for higher liver weights in males treated with 150 mg/kg/day was, in the absence of histopathological changes, considered to represent an adaptive change.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The gross findings noted in this study did not distinguish test item-related rats from control rats.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A number of microscopic findings were considered to distinguished test item-treated rats from the control rats at the end of the treatment period:
- Liver: centrilobular hepatocellular hypertrophy was noted in four of five male and four of five female rats treated with 450 mg/kg/day. the severity of this finding was minimal to slight.
- Thyroid gland: a slightly increased incidence of follicular cell hypertrophy was noted in male and female rats treated with 450 mg/kg/day.
All other findings noted in this study were considered to be incidental findings commonly occurring in rats of this strain and age.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
- Viability/mortality: One female (no. 38) treated with 450 mg/kg/day died on treatment day 11. No clinical signs were evident and the cause of death could not be established. All other animals survived until scheduled necropsy.
- General cage side observations (daily): dark orange bedding, noted from day 9 onwards in the males and females treated with 450 mg/kg/day, was considered to be a test item-related effect possibly caused by an unspecified urinary metabolite. All remaining findings (salivation, kinked tail, localized alopecia) were considered to be incidental findings.
– Detailed clinical observations (weekly): no abnormal clinical signs were noted during treatment week 1, 2 or 3.

BODY WEIGHT AND WEIGHT GAIN
On treatment days 8, 15, 22 and 28, the mean body weights and the mean body weight gain of males treated with 450 mg/kg/day were slightly lower than those of the control males. The difference from the mean body weight gain of the control males which were noted on treatment day 8 attained statistical significance (p<0.05). These changes were considered to be test item-related. The mean body weights and the mean body weight gain of the males treated with 50 mg/kg/day and 150 mg/kg/day were similar to those of the controls. The mean body weights and mean body weight gain of test item-treated females compared
favorably with those of the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The mean daily food consumption of the females treated with 450 mg/kg/day was marginally reduced during the first measurement interval (days 1-8 of treatment), but increased to control levels during the remaining part of the study (day 8-28 of treatment). These differences were reflected in the relative food consumption data. The mean daily food consumption of the test item-treated males and remaining test itemtreated females compared favorably with those of the controls

FOOD EFFICIENCY
not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
not examined

OPHTHALMOSCOPIC EXAMINATION
not examined

HAEMATOLOGY
The hematology parameters of the test item-treated females were similar to those of the controls.
The mean cell hemoglobin and the mean cell hemoglobin concentration of the test itemtreated males were significantly higher (p<0.01) than those of the control males. All values remained within the 95% tolerance limits of the historical control data, and the differences were considered to be unrelated to the test item. The mean cell volume of the males treated with 50 mg/kg/day was also significantly increased (p<0.05) when compared with the controls. In the absence of a dose response-relationship, these changes were considered to be incidental.
The significantly abbreviated thromboplastin times noted in the females treated with 150 mg/kg/day (p<0.05) and 450 mg/kg/day (p<0.01) when compared with the control females remained within the 95% tolerance limits of the historical control data and were considered to be unrelated to the test item.

CLINICAL CHEMISTRY
Significantly increased levels of total cholesterol and phospholipids (p<0.01) were noted in females treated with 450 mg/kg/day. Although these changes remained within the 95% tolerance limits of the historical control values, they were accompanied by significantly higher activities of alanine aminotransferase (p<0.01, exceeding the historical control ranges) and gamma glutamyltransferase (p<0.01). These findings were considered to be evidence of changes in liver metabolism and related to the treatment with the test item.
The significant reductions of lactate dehydrogenase activity noted in females treated with 150 mg/kg/day (p<0.01) and 450 mg/kg/day (p<0.01) were considered not to be test item-related changes.
In males treated with 450 mg/kg/day, significant increases in phosphorus levels (p<0.01) and potassium levels (p<0.01) were noted when compared with the control males. These differences were not seen in females, nor did they exceed the 95% tolerance limits of the historical control data.
All differences in the males treated with 50 mg/kg/day (significantly increased potassium and chloride, p<0.05) were not dose dependent, remained within the 95% tolerance limits of the historical control data and were therefore considered to be incidental.

URINALYSIS
not examined

NEUROBEHAVIOUR
- All findings (salivation, kinked tail, localized alopecia) were considered to be incidental findings and all females were without clinical signs.
- Grip strength: The mean fore- and hindlimb grip strength of the test item-treated rats compared favorably with those of the respective controls.
- Locomotor activity: Significantly reduced locomotor activity was noted during 0-15 minutes in males treated with 450 mg/kg/day (p<0.05), and the overall locomotor activity (0-60 minutes) was significantly reduced (p<0.01) when compared with the control males. In the absence of a similar change in the females (in which a significant increase was noted from 45-60 minutes, p<0.05), these differences were considered to be unrelated to the test item. When compared with the controls, significant differences in the locomotor activity of females treated with 50 mg/kg/day (increased activity from 30-45 minutes and 45-60 minutes) (p<0.05), were not dose-dependent and therefore considered to be incidental.

ORGAN WEIGHTS
Significantly increased liver weights were noted in males (p<0.05) and females (p<0.01) treated with the test item at 450 mg/kg/day when compared with the controls. In males, the liver-to-body weight ratio was significantly higher at 150 mg/kg/day (p<0.05) and 450 mg/kg/day (p<0.01) and the liver-to-brain weight ratio was significantly higher at 450 mg/kg/day (p<0.01). In females, both relative liver weights were increased significantly (p<0.01) when compared with the controls. Increased liver weights were considered to be test item-related changes.
The trend for higher liver weights in males treated with 150 mg/kg/day was, in the absence of histopathological changes, considered to represent an adaptive change.
The remaining organ weights were considered to be similar to the control values

GROSS PATHOLOGY
There were no gross findings considered to distinguish test item-treated rats from the control rats.

HISTOPATHOLOGY: NON-NEOPLASTIC
A number of findings were noted at the end of the treatment period. From these findings, the following ones distinguished test item-treated rats from the control rats:
- Liver: centrilobular hepatocellular hypertrophy was noted in four of five male and four of five female rats treated with 450 mg/kg/day. The severity of this finding was minimal to slight.
- Thyroid gland: a slightly increased incidence of follicular cell hypertrophy was noted in male and female rats treated with 450 mg/kg/day.
A number of findings were diagnosed in the other organs and tissues examined at the termination of the treatment period. Their incidence, severity and morphologic appearance did not distinguish test item-treated rats from the control rats.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
not applicable

HISTORICAL CONTROL DATA (if applicable)
Hematology and clinical chemistry data were compared to the historical control data.


OTHER FINDINGS
no data

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: No test substance-related adverse effects were observed at 50 mg/kg/day dose level in both males and females.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased liver weights noted in males treated with 150 mg/kg/day. Thes were considered to be adaptive changes since no histopathological changes were evident.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Determination of Concentration, Homogeneity, and Stability of TOO2113 in Bi-distilled Water

Acclimatization/Pretest:

The mean concentrations of the homogeneity samples were found to be 101.8%, 110.0%, and 110.6% of the nominal concentrations of dose group 2 (5 mg/ml), dose group 3 (15 mg/ml), and dose group 4 (45 mg/ml), respectively. The individual concentrations varied

in the range from -5% to +7% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle. T002113 is stable in the vehicle under storage conditions for at least seven days.

Administration/Test:

The overall mean concentrations of the homogeneity samples were found to be 102.7%, 103.7%, and 105.2% of the nominal concentrations of dose group 2 (5 mg/ml), dose group 3 (15 mg/ml), and dose group 4 (45 mg/ml), respectively. The individual concentrations varied in the range from -1 % to + 1% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Applicant's summary and conclusion

Conclusions:
Oral administration of T002113 to Wistar rats at doses of 50, 150 and 450 mg/kg/day, for 28 days resulted in no clinical signs of toxicological relevance (daily, weekly or functional observational battery), no effects upon fore- and hindlimb grip strength or locomotor activity, no changes in hematology parameters and no macroscopical changes related to the administration of the test item.
The single death (female no 38 treated with 450 mg/kg/day) was not accompanied by overt clinical signs nor was the cause of death apparent at histopathological examination.
Test item-related findings of toxicological relevance were generally restricted to animals treated with 450 mg/kg/day, and included dark orange discoloration of the bedding, a transient reduction of food consumption in females, slightly lower body weights in males, changes in clinical biochemistry parameters in females indicative of effects upon liver metabolism, increased liver weights in males and females, minimal to slight centrilobular hepatocellular hypertrophy was noted in males and females, and a slightly increased incidence of follicular cell hypertrophy of the thyroid in males and females.
Increased liver weights, noted in males treated with 150 mg/kg/day were considered to be adaptive changes since no histopathological changes were evident.
Based on the results of this study, 50 mg/kg body weight/day of T002113 was established as the no-observed-effect-Ievel (NOEL) and 150 mg/kg/day was considered to be the no-observed-adverse-effect-Ievel (NOAEL).