4-[(6-chloro-2-[(4-cyanophenyl)amino]-4-pyrimidinyl]oxy]-3,5-dimethylbenzonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-01-21 to 2003-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-({6-chloro-2-[(4-cyanophenyl)amino]pyrimidin-4-yl}oxy)-3,5-dimethylbenzonitrile

EC Number:

928-806-6

Cas Number:

1070377-34-2

Molecular formula:

C20H14ClN5O

IUPAC Name:

4-({6-chloro-2-[(4-cyanophenyl)amino]pyrimidin-4-yl}oxy)-3,5-dimethylbenzonitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): T002615
- Physical state: solid
- Appearance white solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): T002615
- Substance type: not applicable
- Physical state: white solid
- Analytical purity: > 98%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 00403487
- Expiration date of the lot/batch: 2003-12-01
- Stability under test conditions: stable under storage conditions
- Storage condition of test material: at room temperature (17-23 deg C) in the original container away from direct sunlight
- Other: no data

Test animals

Species:

rat

Strain:

other: HanBrl:Wist (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd ,Biotechnology & Animal Breeding Division CH-4414 Fullinsdorf/Switzerland
- Age at study initiation: 6 weeks at delivery
- Weight at study initiation: Males: 140.8 -159.7 g ( mean 150.0 g); Females: 117.0-132.3 g (mean 123.5 g)
- Fasting period before study: no
- Housing: in groups of five rats in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding (Lignocel Schil AG, CH-4132 Muttenz/Switzerland)
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3433 (batch no.90/02 rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland ) ad libitum
- Water (e.g. ad libitum): community tap water from Itingen ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 (+/- 3 deg C)
- Humidity (%): 30-70%
- Air changes (per hr):10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs fluorescent light /12 hrs dark (miusic played during the light period)

IN-LIFE DATES: From: 2003-01-21 To:2003-03-04

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: PEG 300

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly. T002615 was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23 deg C). Homogeneity of the test item in the vehicle wasmaintained during the daily administration period using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency):no data
- Mixing appropriate amounts with (Type of food):no data
- Storage temperature of food: no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): 442989/ 1 54802033
- 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. 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 an HPLC method supplied by the Janssen Pharmaceutica N.V.
The test item was found to be homogeneously distributed in the vehicle.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Basis: based upon the results of a non-GLP 5-day dose- range finding study in which T002615 was administrated by gavage to 2 rats per group and sex

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Basis: based upon the results of a non-GLP 5-day dose- range finding study in which T002615 was administrated by gavage to 2 rats per group and sex

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Basis: based upon the results of a non-GLP 5-day dose- range finding study in which T002615 was administrated by gavage to 2 rats per group and sex

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Basis: based upon the results of a non-GLP 5-day dose- range finding study in which T002615 was administrated by gavage to 2 rats per group and sex

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 in which T002615 was administrated by gavage to 2 rats per group and sex
- Rationale for animal assignment (if not random): randomization: computer-generated random algorithm
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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 4-28
- Cage side observations included: mortality/viability, general clinical observations

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 observation were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:weekly during pretest, treatment and before necroscopy, using an online electronic recording system of a Mettler balance connected to the RCC computer

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- Food consumption for each animal determined and mean daily diet 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: No

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): not applicable

OPHTHALMOSCOPIC EXAMINATION: not applicable

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (18 hrs before blood sampling)
- How many animals: all animals
- 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, total leukocyte count, differential leukocyte count, coagulation: thromboplastin time, and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes (18 hrs before blood sampling)
- How many animals: all animals
- Parameters examined: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: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all groups
- Battery of functions tested: grip strength and motor activity

OTHER: observation for mortality/viability were recorded twice daily

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
-Description of all macroscopic abnormalities were recorded
HISTOPATHOLOGY: Yes
All animals 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 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 (including 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 (including parathyroid gland), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina, gross lesions. All organ and tissue samples were processed, embedded and cut at an app. thickness of 2 to 4 micrometers, and stained with hematoxylin by a pathologist.

Other examinations:

The following organ weights were recorded on the scheduled dates of necroscopy: 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 necroscopy.

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 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
-Armitage/Cochran Trend test was used for non-neoplastic lesion

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were noted during daily or weekly observation (weeks 1-3).
-general cageside observation (daily): slightly soft feces was seen in all test item treated animals and controls treatment day 19 to the end of treatment. This was due to oral gavage of polyethylene glycol 300. Slightly pale feces was noted from treatment day 10 until the end of treatment in all animals treated with 200 mg/kg/day or 1000 mg/kg/day of the test item. This was considered to be passive effect of the test item. Slight alopecia on the head was seen between treatment days 10 and 21 and slight crusts on the head was seen between days 10 and 16 in one female treated with 50 mg/kg/day of the test item. This was isolated finding and considered to be incidential. No other clinical signs were noted during daily observations.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necroscopy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight of males treated with 200 mg/kg/day was increased (p<0.05) in treatment week two when compared to controls. this was considered to be incidential because no dose response relationship could observed and it was not seen after three or four weeks of treatment. No other difference in mean body weight or body weight gain were noted in test item treated males/females when compared with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No difference in absolute or relative food consumption were noted in males or females when compared with controls.

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):

The mean activated partial thromboplastin time was prolonged (p< 0.05) after four weeks of treatment in females treated with 1000 mg/kg/day. This was considered to be incidental because the value lies within the range of the historical control data. No other statistically significant changes in parameters of hematology were seen in males and females when compared with controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

-The mean inorganic phosphate level was decreased (p<0.01) after four weeks of treatment in males treated with 50 mg/kg/day, and the mean sodium level was increased (p<0.05) in males at 1000 mg/kg/day when compared with controls. These findings were considered to be incidential because no dose response relationship could be observed and it was not seen in females.
-The mean level of albumin was increased in males treated with 50mg/kg/day when compared with controls. This was considered to be incidential because no dose response relationship could be observed.
-No other statistically significant changes in parameters of clinical biochemistry were seen in males/females when compared with controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in parameters were noted during the functional observational battery (during weekly observation, week 4)
Grip strenght: decreased (p<0.05) mean forelimb grip strength was observed in females treated with 50 mg/kg/dat of the test item when compared with controls. This dose-unrelated finding was considered to be incidental. No other differences in mean fore- or hindilimb grip strength were observed in test item treated animals when compared with controls.
Locomotor activity: the mean locomotor activity was decreased (p<0.01) in males treated with 200 mg/kg/day between 30 and 40 minutes of the one hour observation period. This was considered to be incidential because it was an isolated finding, unrelated to dose. No other changes in mean locomotor activity were observed in test item treated animals when compared with controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related changes in mean organ weights, organ-to -body weight or organ-to-brain weight ratios were observed in males/females after four weeks of treatement when compared with controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

-Macroscopic findings: at the end of the treatment the following gross lesions were detected: splenic constriction in one animal, several foci in the thymus of one animal ( female, 50 mg/kg/day). These findings were considered to be typical background changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

-Stomach (microscopic finding): the test item , T002615 produced minimal to slight signs of topical irrative effects in the stomach in animals dosed with 1000 mg/kg/day, less pronounced , in animals dosed with 200 mg/kg/day of the test item. These findings consisted of minimal to slight grades of hyperkeratosis, epithelial hyperplasia and basal cell hyperplasia of the limiting ridge. There was also a tendency to increased submucosal inflammatory infiltrates.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

1

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of this study , 50 mg/kg bw/day of T002615 was established as the no-observed-effect-level (NOEL) and >=1000 mg/kg bw/day of T002615 as the no-observed-adverse-effect-level (NOAEL).