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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:
07 December 1999 - 04 January 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-380-1
EC Name:
-
Cas Number:
133336-92-2
Molecular formula:
C29H28N4O2
IUPAC Name:
1-(4-methylphenyl)-3-{4-[(4-{[(4-methylphenyl)carbamoyl]amino}phenyl)methyl]phenyl}urea
Test material form:
solid: particulate/powder
Details on test material:
- Particle size distribution: 10%: < 23.94 µm
- Density: 1.32 g/cm3 (20°C)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:(WI) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males: 176 - 202 g; females: 154 - 175 g
- Fasting period before study: No data.
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, animals were individually housed overnight in Macrolon plastic cages.
- Diet: Free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium)
- Water: Free access to tap-water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 December 1999 To: 03 January 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing. Adjustment was made for specific gravity of vehicle (1.036).

DOSE VOLUME: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis:
Samples of week 1 formulations were analysed to check stability over 4 hours, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days/week.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study. No mortality and no clinical signs were observed in the 50, 150 and 1000 mg/kg bw/day groups. Based on the results, dose levels for the main study were selected to be 50, 150 and 1000 mg/kg bw/day.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily. Once prior to start of treatment and on days 8, 15, 22 and 28.

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28.

FOOD CONSUMPTION
- Weekly.

WATER CONSUMPTION
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, functional observations:
During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength
- motor activity test (recording period: 12 hours during overnight for individual animals)

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

NECROPSY:
All animals surviving to the end of the observation period (day 29) were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.

ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes and Thymus.

HISTOPATHOLOGY: Yes
HISTOTECHNOLOGY
All organ and tissue samples (according to the guidelines), as defined under Histopathology (following), were processed, embed ded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions of all animals were examined by a pathologist.
Based on the treatment related morphologic changes, the stomach was also examined from all rats of the intermediate dose groups. All abnormalities were described and included in the report.
Other examinations:
HAEMATOLOGY: Met-haemoglobin
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.30 and 9.30 a.m.
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all rats/sex/group
Statistics:
The following statistical methods were used to analyse the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test will be applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores). Test statistics were calculated on the basis of exact values for means and pooled variances.

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):
not examined
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The levels of met-haemoglobin showed an dose related increase and achieved levels of statistical significance (p<0.01) in both high dose males and females, when compared with controls. The group-mean values of met-haemoglobin for the animals of the mid dose groups did not achieve levels of statistical significance. As these values were within the range of control values, the slight increase in the mid dose groups was considered of no toxicological significance.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
Clinicals signs:
Incidental findings that were noted among treated and control animals included a wound (neck), scabs (neck), alopecia (neck, back and flanks) and salivation. These findings are commonly noted in rats of this age and strain and housed and treated under the conditions in this study and considered of no toxicological significance.

Food consumption:
The food consumption of the substance treated females showed a slight decrease at higher dose levels. In the absence of clear signs of toxicity in the females, no toxicological significance was attached to this effect. The food consumption of test substance treated males was considered to be similar to that of controls.

Haematology:
A few other haematological parameters achieved levels of statistical significance in high dose females only, comprising the erythrocyte indices, mean corpuscular volume and mean corpuscular haemoglobin, platelets and relative number of monocytes.
Based on the absence of changes in the number of erythrocytes and haemoglobin in these animals to support the changes in the erythrocyte indices and the fact that the statistical significance was only minor (p<0.05), no toxicological significance was attached to these findings.
In the absence of a clear dose response relationship and since all values are within the range of historical background data, the toxicological significance of the increased number of platelets and monocytes was doubted.
The minor statistically significant differences for white blood cells (low dose males) and platelets (low dose females) were considered to have arisen by chance. In the absence of a dose-response realtionship, no toxicological significance was attached to these findings.

Clinical Biochemistry:
In high dose males, increased levels of alanine-and aspartate-aminotransferase were found, achieving levels of statistical significance when compared to controls. Since the increases were relatively minor and were not supported by changes in liver weight and after microscopic examination of the liver, no toxicological significance was attached to these findings.
The statistical significance for the levels of urea (high dose males, p<0.05), alkaline phosphatase (low dose males, p<0.05) and inorganic phosphate (mid dose females, p<0.05) were considered to have arisen by chance, and of no toxicological significance.

Macroscopic examination:
The scab-formation seen in the neck of a high dose male confirmed the clinical signs in this animals seen during the in-life phase, and was considered a change of no toxicological significance.

Organ weights:
The statistical significance arising for the absolute and relative heart weights of high dose females was the result of a sligtly low heart weight in controls and considered to have arisen by change.
Organ weights and organ: body weight ratios of treated males were considered to be similar to those of controls.
In the absence of a dose response relationship, no toxicological significance was atatched to the minor statistically significant changes between absolute and relative thymus weights and of absolute adrenal weights in low dose females.

Functional observations:
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with the substance, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.

Analysis of dose preparations:
Analysis of the accuracy of dose preparations revealed values of approximately 90% for the high and mid dose formulations and of approximately 100% for the low dose formulation.
It should be noted that the analysed concentrations of the mid and high dose were at the border of the range of acceptability for this type of preparations (NOTOX criteria: maximum deviation s10% from prepared). The relatively low recovery in the mid and high dose preparations may be attributed to the poor solubility of the test substance in the (analytical) solvents used. Since an acceptable recovery was seen in the low dose preparation and the raw data for all dose preparations showed that the correct amounts of test substance were weighed, all dose preparations were accepted for use in this study.
The test substance preparations in propylene glycol at the concentrations tested, were considered to be homogeneous and sufficiently stable (over 4 hours) to be used in these types of studies.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
other: Formation of Met-Hb
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
In an 28-day repeated dose oral toxicity study in rats performed according to OECD 407 guideline and GLP principles, the NOAEL was determined to be 150 mg/kg bw/day.
Executive summary:

An 28-day repeated dose oral toxicity study in rats was performed with KY-AF according to OECD407 guideline and GLP principles. Based on the results of a 5-day dose range finding study, dose levels

for the main study were selected to be 50, 150 and 1000 mg/kg bw/day. No mortality occurred during thestudy period. There were no changes in clinical appearance, functional observations, body weights, food consumption, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment.

The levels of met-haemoglobin showed an dose related increase and achieved levels of statistical significance (p<0.01) in both high dose (1000 mg/kg bw/day) males and females, when compared with controls. The group-mean values of met-haemoglobin for the animals of the mid dose groups did not achieve levels of statistical significance. As these values were within the range of control values, the slight increase in the mid dose groups was considered of no toxicological significance.

Based on the results of the study, the NOAEL was determined to be 150 mg/kg bw/day.