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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:
2005-10-20 to 2006-03-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD guideline 407 and EC method B7 without deviations.

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:
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)
Deviations:
no
GLP compliance:
yes
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:
- Name of test material (as cited in study report): TIC2782 (T002907)
- Substance type: White solid
- Physical state: solid
- Analytical purity: 99.9%
- Purity test date: No data
- Lot/batch No.: 00467090 (=charge 05C0836)
- Expiration date of the lot/batch: 2006-05-04
- Stability of test item: Stable under storage conditions
- Storage condition of test material: at room temperature (15-25°C) light-protected

Test animals

Species:
rat
Strain:
other: 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
- Age at delivery: 6 weeks
- Weight at study initiation: males: 140 - 153 gr (mean 148 gr); females: 116-134 gr (mean 126 gr)
- Fasting period before study: No data
- 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 (e.g. ad libitum): Ad libitum, pelletted standard Provimi Kliba 3433 rat maintance diet
- Water (e.g. ad libitum): Ad libitum, community tap-water from Itingen in water bottles.
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. Under test conditions after health examination. Only animals without any visible signs of illness were used for this study

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

IN-LIFE DATES: From: 2005-10-31 To: 2005-11-28

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The dose formulations were prepared weekly. TIC2782 (T002907) was weighed into a glass beaker on a tared Mettler Balance and the vehicle added. THe mixtures were prepared using a homogenizer to reducte the granular structure of the test item, followed by magnetic stirrer and stored at room temperature. 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 data
- Amount of vehicle (if gavage): 5mL/kg body weight
- Lot/batch no. (if required): 1143422 35004049 (from 2005-10-31 until 2005-11-06) and 1195971 13505272 (from 2005-11-07 until 2005-11-27)
- Purity:No data
- Storage conditions: At room temperature (15- 25 °C) away from direct sunlight.
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. The analyses were performed by the Principal Invesitgator of the analytica phase (RRC, Ltd, Environmental Chemistry & Pharmanalytics DIvision) accoriding to a GC method supplied by the sponsor. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
oral by gavage
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
oral by gavage
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
oral by gavage
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
oral by gavage
No. of animals per sex per dose:
5
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 A28754) in which TIC 2782 (T002907) was administrated by gavage to 2 rats per group and sex.
- Rationale for animal assignment (if not random): computer-generated random algorithm

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality/viability: twice daily; general cageside observations: daily, 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:weekly: 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 acclimatization, treatment and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

ABSOLUTE AND RELATIVE 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.

FOOD CONSUMPTION
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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION:No

HAEMATOLOGY: Yes
Blood sampling: after 4 weeks: 28 November 2005. Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted 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 rhytms. Blood samples were drawn from the recto-orbital plexus using a micro-hematocrit glass capillary tube.
- The following parameters were determined: 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, total leukocyte count, differential leukocyte count, coagulation, thromboplastin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
Blood sampling: after 4 weeks: 28 November 2005. Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted 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 rhytms. Blood samples were drawn from the recto-orbital plexus using a micro-hematocrit glass capillary tube.
- The following parameters were determined: glucose, urea, creatinine, bilirubin total, cholesterol total, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, triglycerides, phospolipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatinine kinase, calcium, phosphorus inorganic, protein total, albumin, globulin, albumin/globulin ratio

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes
- 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. Sacrifice: after 4 weeks on 28 November 2005. All animals were weighed and necropsied. 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 (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 soltuion), 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 Bouin's solution), thymus, thyroid (incl. parathyroid gland), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina, gross lesions.

ORGAN WEIGHTS
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 which were collected at scheduled sacrifice from animals of control and high-dose groups were examined by a pathologist.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, macroscopical findings, 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 can not be assumed to follow a normal distribution.
- Fischer's exact-test was applied to the macroscopic findings.
- Student's T-test was applied to locomotor activity and grip strength.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived until scheduled necropsy. No clinical signs of toxicological relevance were noted during the daily cageside observations in test item-treated rats of both sexes at any dose level. No clinical signs of toxicological relevance were noted durign the weekly observations in test item-treated rats of both sexes at any dose level.

BODY WEIGHT AND WEIGHT GAIN
No test item-related changes of toxicological relevance were noted in body weight and body weight gain of male and female rats at any dose level during the four-week treatment period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean daily- and relative food consumption of test item-treated rats compared favorably with their respective controls at all dose levels during the four-week treatment period.

HAEMATOLOGY
No changes of toxicological relevance were noted in the hematology paramaeters after the treatment period in test item-treated rats of both sexes. In males treated with 200 mg/kg/day, decreased hemoglobin (HB, p<0.05) and decreased hematocrit (HCT, p<0.05) were noted. As no dose response relationship could be established and as the values remained within the range of the historical control data, these findings were considered to be incidental.

CLINICAL CHEMISTRY
No changes of toxicological relevance were noted in the clinical biochemistry parameters after the treatment period in the test item-treated rats of both sexes. Minor changes remaining within the range of historical control data were noted in female rats treated with 50 mg/kg/day or with 200 mg/kg/day and included increased sodium concentrations (both p<0.05). These findings were therefore considered to be fortuitous.

NEUROBEHAVIOUR
No toxicologically relevant test item-related changes were noted in mean fore- and hind limb grip strength of male and female rats.
-Grip strength: In male rats treated with 200 mg/kg/day, the hind limb grip strength was significantly (p<0.05) increased when compared to control males. As no dose response relationship could be established, ths observation was considered to be fortuitous.
-Locomotor acitvity: Increased locomotor activity was recorded in males treated with 50 mg/kg/day (p<0.05) and in rats of both sexes treated with 200 mg/kg/day (both p<0.05). Increased locomotor activity in females treated with 50 mg/kg/day or with 200 mg/kg/day was noted during the 30-40 minutes measurement interval (both p<0.05). As no dose response relationship could be established, all these changes were considered to be incidental.

ORGAN WEIGHTS
Male rats treated with 1000 mg/kg/day presented decreased mean absolute testes weight (p<0.05) after a four-week treatment period. In females treated with 1000 mg/kg/day, decreased mean absolute adrenals weight (p<0.05) and increased thymus-to body weight ratio (both p<0.05) were noted. These statistically significant deviations from their respective controls were considered to be incidental, reflecting usual indvidual variability and therefore lacking toxicological relevance.

GROSS PATHOLOGY
At the end of the treatment period no test item-related gross lesions were observed. The macroscopic findings recorded were considered to be within the range of normal background lesions, which may be seen in species of this strain and age in this study type and were considered incidental, reflecting the usual individual variability.

HISTOPATHOLOGY: NON-NEOPLASTIC
A variety of changes were found in this study. They commonly occur in laboratory rats of this strain and age, neither their incidences nor their distribution or morphological characteristics gave any indication of a test item-related association.

Effect levels

open allclose all
Dose descriptor:
NOEL
Based on:
test mat.
Basis for effect level:
other: Based on the results of this study, the NOEL could not be established
Remarks on result:
not measured/tested
Remarks:
Effect level not specified
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Results of analytical verification:

This chemical analysis determined concentration, homogeneity, and stability of T002907 in PEG300.

-date of preparation: 24 -OCT-05

The mean concentrations of the homogeneity samples were found to be 101.6%, 99.8%, and 102.1% of the nominal concentrations of dose group 2 (10.0 mg/ml), dose group 3 (40.0 mg/ml), and dose group 4 (200.0 mg/ml), respectively. The individual concentrations varied in the range from -3% to +2% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

-date of preparation: 14 -NOV-05

The mean concentrations of the homogeneity samples were found to be 101.7%, 95.0.%, and 97.0% of the nominal concentrations of dose group 2 (10.0 mg/ml), dose group 3 (40.0 mg/ml), and dose group 4 (200.0 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.

T002907 is stable in the vehicle under storage conditions for seven days.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed-effect level (NOEL) could not be established but 1000 mg/kg bodyweight/day of TIC2782 (T002907) was established as the no-observed-adverse-effect-level (NOAEL).