2-(chloromethyl)-4-methylquinazoline

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:

2007-01-22 to 2007-02-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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)

Version / remarks:

1995

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

476-280-7

EC Name:

-

Cas Number:

109113-72-6

Molecular formula:

C10 H9 Cl N2

IUPAC Name:

2-(chloromethyl)-4-methylquinazoline

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: Off-white with yellow spherical particles

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charkes River (Europe), Laboratories Inc. Toxi Coop Ltd. 1103, Budapest, Cserkesz u. 90.
- Age at study initiation: 7 weeks old
- Weight at study initiation: male: 203 – 216 g ; female: 170 – 185 g
- Housing: 5 animals/cage; cage type: Type III. polypropylene/polycarbonate
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 8-12 air exchanges/hour by central air-conditioning system
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6:00 a.m. to 6:00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % aqueous methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared in 1 % aqueous methylcellulose in concentrations of 2 mg/L, 10 mg/L and 25 mg/L daily or stored for no longer than for 48 hours in the Central Dispensary of LAB International Hungary Ltd. Analytical control of samples was performed in the Analytical Laboratory of LAB international Hungary Ltd. during the first and last weeks of the study. The concentration, homogeneity and stability of dosing solutions were checked. CD 605 was stable in concentrations of 1 mg/mL and 100 mg/mL in this vehicle at room temperature for 24 hours and in the refrigerator for 48 hours. Measured samples were homogenous and concentrations were close to nominal concentrations (deviation is 96-109 % of the nominal values) in the chosen vehicle for the different treatment solutions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical method:
HPLC system: Merck Hitachi LaChrom HPLC system
Detector: UV at 230 nm
Column: Zorbax Eclipse XDB-C18 150 mm*4.6, 5 µ
Mobile Phase: Acetonitrile/water = 4/6 (v/v)
Flow: 1 ml/min
Injection volume: 10 µl
Column Temperature: 40°C
Retention time: 4.0 +- 10%

Results of the Method Validation:
Repeatability (Instrument precision) <= 1%
Linear range: 0.02 - 10 µg/ml
LOD: 0.01 µg/ml
LOQ: 0.02 µg/ml
Recovery: from methyl cellulose suspension: 101 and 106 %
from ultra-pure water: 98 %
Accuracy: 0-7 %
Precision: 1-5 %

Stability: Test item proved to be stable for at least 14 days in stock solution at 5 +- 3°C.
34 hours in the auto sampler, dissolved in the mobile phase
24 hours in methyl cellulose suspension at 20°C
48 hours in methyl cellulose suspension in refrigerator

Standard Solutions: 7 calibration solutions were diluted with the mobile phase. Concentrations were: 0.02, 0.05, 0.1, 0.5, 1, 5 and 10 µg/ml.

The peak area was plotted against concentration and a regression analysis yielding the calibration curve
Constant X Coefficient R Square
Calibration curve 1: 278 103141 1.000
Calibration curve 2: 221 102611 1.000

Analysis of Samples:
Nine samples were taken from each dosing formulation (3 samples from lower, middle and upper parts of the vessels). One sample was taken from the control sample suspension.

Measured values at the first week of the test
Nominal concentration (mg/ml) Measured concentration +- confidence interval 95% (mg/ml) Measured/Nominal concentration %
2 2.05 +- 0.03 102
10 10.0 +- 0.2 100
25 26.0 +- 0.7 104

Measured values at the last week of the test
Nominal concentration (mg/ml) Measured concentration +- confidence interval 95% (mg/ml) Measured/Nominal concentration %
2 1.93 +- 0.07 96
10 10.6 +- 0.4 106
25 27.2 +- 0.8 109
Concentration and homogeneity of the dosing formulations were determined during the first and at the last week of the study. CD 605 was not detected in the control sample. All dosing formulations were homogeneous, the measured test item concentrations are given in table below

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day (7-days per week)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals/sex/ group (4 groups)
=20 males, 20 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of a preliminary dose-finding study
- Rationale for animal assignment: random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A general clinical examination was made once a day at approximately the same time. Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).

BODY WEIGHT: Yes
- Time schedule for examinations: For each animal body weight was recorded on day 0 (beginning of the experiment), then weekly with precision of 1g. On the day of gross pathology, fasted body weights were determined.

FOOD CONSUMPTION:
- The food consumption was determined by re-weighing the non-consumed diet weekly with a precision of 1 g.

FOOD EFFICIENCY:
- Body weight gain in g: Yes

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes (Euthanyl)
- Animals fasted: Yes (approximately 16 hours)
- How many animals: each animal
- Parameters checked in table [No.3] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes (approximately 16 hours)
- How many animals: each animal
- Parameters checked in table [No.3] were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathology was performed one day after the last treatment. Animals were sacrificed following blood sampling. After exsanguination the external appearance was examined. Cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size.


HISTOPATHOLOGY: Yes
The following organs/tissues were removed and preserved in 10 % buffered formaldehyde solution for histology processing:
Gross lesions, lymph nodes (submandibular, mesenteric), sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, testes with epididymitis, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, Harderian
glands and lachrymal gland, seminal vesicle, muscle (quadriceps), sciatic nerve, aorta.

Statistics:

The following statistical algorithms were used:
Bartlett's variance test;
Duncan's Multiple Range test;
Kolmogorov-Smirnov test;
Kruskal-Wallis One-Way analysis of variance

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no significant effects

Mortality:

mortality observed, treatment-related

Description (incidence):

no significant effects

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

no significant effects

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

no significant effects

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 RDW% and Reticulocyte count in the female animals as well as the Prothrombin time in males of the high dose groups showed differences compared to control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related elevated calcium concentration at 100 mg/kg bw/day and 250 mg/kg bw/day; the mean protein, albumin and total bilirubin levels were higher than control in the male animals at the highest dose.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The slightly elevated spleen and liver weight at 250 mg/kg bw/day females were indication of functional changes.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

no significant effects

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

no significant effects

Details on results:

MORTALITY
One female animal was found dead on day 9. No clinical symptoms were noted before the death. Gross pathology revealed dark red lungs. The death was not ascribed to the test item. The alteration could be consistent with an acute circulatory insufficiency.

BODY WEIGHT AND WEIGHT GAIN
Groups 20 mg/kg bw/day, 100 mg/kg bw/day and 250 mg/kg bw/day
The body weight and body weight gain were similar to those of control group in all test item treated groups (male and female) throughout the 28-day period. An exception was observed in the female 100 mg/kg bw/day and 250 mg/kg bw/day dose groups, where body weight gain was slightly above the control value on week 4. However this observation was not considered toxicologically significant.

HAEMATOLOGY
Most of the statistical differences between groups were within the historic control range and had no biological significance. The only haematology parameters, in the context of historical control data, that indicated of a possible treatment related effect were the higher RDW% and Reticulocyte count in the female high dose group and shorter Prothrombin time in the male high dose group. For Reticulocyte count and Prothrombin time, a statistically significant difference was seen in both males and females, although each was only outside the historic range in one sex.
The differences were relatively small in biological terms. The higher Reticulocyte count usually indicates a higher turnover of erythrocytes, the higher RDW% would support this observation. But in this study there was no indication of anaemia, so any effect on erythrocytes was fully
compensated. The slightly shorter Prothrombin time was a small difference in males, and the statistical difference in females was due to unusually high control values. Taking into account the overall profile, the difference in Prothrombin time is considered to be of little or no biological significance.

CLINICAL CHEMISTRY
A dose related higher calcium concentration was observed in both sexes at 100 and 250 mg/kg bw/day. In males dosed at 250 mg/kg bw/day the mean protein, albumin and bilirubin levels were slightly but significantly higher than controls. Other statistical differences in clinical chemistry parameters were not considered to be of biological significance because either the control values were lower than historical control levels, or the differences were small and all values were within the historic control range.

ORGAN WEIGHTS
The statistically significantly higher liver and spleen weights in females in group 4 were considered a test item related effect, however, the observations were not supported by histopathological findings. Values were in the normal range, so there was no indication of a significant toxic effect.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

2

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The test item induced elevated calcium concentration in both sexes of Wistar rats at 100 mg/kg bw/day and 250 mg/kg bw/day after 28-day continuous oral (by gavage) administration. The mean protein, albumin and total bilirubin levels were higher than control in the male animals at the highest dose.

The RDW% and Reticulocyte count in the female animals as well as the Prothrombin time in the males of the high dose groups showed differences compared to the control. The slightly elevated spleen and liver weights at 250 mg/kg bw/day in females without any histopathological findings were an indication of functional changes.

Applicant's summary and conclusion

Conclusions:

The no observed effect level (NOEL) was 20 mg/kg bw/day.
The no observed adverse effect level (NOAEL) was 100 mg/kg bw/day.