5-amino-6-methyl-1,3-dihydrobenzoimidazol-2-one

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:

1998-06-08 - 1998-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

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:

adopted 27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted September 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-410-9

EC Name:

-

Cas Number:

67014-36-2

Molecular formula:

C8 H9 N3 O

IUPAC Name:

5-amino-6-methyl-2,3-dihydro-1H-1,3-benzodiazol-2-one

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks
- Housing: group housing of 5 animals per sex
- Diet: standard pelleted laboratory animal diet, ad libitum (Carfil Quality BVBA, Oud-Turnhout, Belgium)
- Water: tap water, ad libitum
- Acclimation period: at least 5 days before treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

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.

VEHICLE
- Justification for use and choice of vehicle: Due to trial formulations performed at NOTOX B.V.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were analysed using HPLC/UV. Test substance formulations in propylene glycol were noted as stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 98% to 104% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.
Accuracy test: All formulations prepared on 10-06-98 were analysed for test substance concentration.
Homogeneity test: The formulations of groups 2 and 4 prepared on 10-06-98 were tested for homogeneity.
Stability test: The formulations of groups 2 and 4 prepared on 10-06-98 were analysed immediately after preparation and after 4 hours storage at ambient temperature.
Samples were analysed using HPLC/UV.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 3, 15, 75 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: Yes
- Food consumption for each animal determined: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to post mortem examination
- Anaesthetic used for blood collection: Yes (ether vapour)
- Animals fasted: Yes
- How many animals: all
- The following Parameters were examined: Erythrocyte count/RBC, Haemoglobin/HB, Haematocrit/HCT, Mean corpuscular volume/MCV, Mean corpuscular haemoglobin/MCH, Mean corpuscular haemoglobin concentration/MCHC, Platelet count, Red cell distribution width/RDW, Total leucocyte count/WBC, Differential leucocyte count/SEG (Neutrophils), EO (Eosinophils), BASO (Basophils), LYMPH (Lymphocytes), MONO (Monocytes)Prothrombin time/PT, Partial thromboplastin time/PTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to post mortem examination
- Animals fasted: Yes
- How many animals: all
- The following Parameters were examined: Alanine aminotransferase/(ALAT/GPT), Aspartate aminotransferase/(ASAT/GOT), Bilirubin (total/BILI T), Cholesterol (total/CHOLEST.T), Creatinine, Glucose, Urea, Protein (total/PROTEIN T), Protein (albumin/ALBUMIN), Alkaline phosphatase/ALP, Sodium, Potassium, Chloride, Calcium, Phosphorus/INORG. PHOSPH

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

Sacrifice and pathology:

GROSS PATHOLOGY
All animals surviving to the end of the observation period were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a 4% formaldehyde solution: Adrenal glands, Aorta, Brain, Caecum, (Cervix), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions

HISTOPATHOLOGY: Yes with all the above collected organs.

Statistics:

The following statistical methods were used to analyse the data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- 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-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
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) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. Incidental findings that were noted among treated and control animals included alopecia, red or brown staining of various parts of the body, a wound, scabs. The incidence of these findings remained within the range expected for group housed rats used in this type of study and were considered of no toxicological significance. The maculate erythema on the tail, noted in all females of the low dose group during the first week of the study, was considered to be caused by biting among the animals. Since this finding was present prior to commencement of treatment it was not taken into account for interpretation. Salivation was observed among control and treated animals. This sign is often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or the taste of the test substance. Therefore, this finding was considered not to be a sign of systemic toxicity.

BODY WEIGHT AND WEIGHT GAIN
Reduced body weight gain, and to a lesser degree lower body weights, were observed in the females receiving 75 mg/kg bw/day from day 15 onwards. Statistically significant mean values for body weight gain were achieved on days 22 and 28 for these animals, when compared to control group values. Over the last week of treatment, the average body weight gain for the females receiving 15 mg/kg bw/day was also slightly lower then of controls (not statistically significant). Body weights and body weight gain of treated males remained in the same range as controls over the 4-week study period.

FOOD CONSUMPTION
There were no marked differences (< 10%) in food consumption before or after allowance for body weight between treated and control animals.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment. The statistically significant difference in differential monocytes count arising between controls and males receiving 15 mg/kg bw/day was considered to have arisen by chance based on the absence of a dose response relationship.

CLINICAL CHEMISTRY
There were no differences noted between control and treated rats that were considered to be related to treatment with the test substance. The decreased values for alanine aminotransferase (ALAT) and aspartate aminotransferase (ALAT) activity, achieving a level of statistical significance in males and females receiving 15 mg/kg bw/day, respectively, were considered of no toxicological significance, based on the absence of a treatment-related distribution or corroborative findings in the opposite sex. Moreover, a decrease in the enzyme levels ALAT and ASAT is considered to be of no biological relevance. The minor statistically significant difference in plasma chloride concentration arising between controls and females receiving 75 mg/kg bw/day was considered to have arisen as a result of a slightly high control value (and small standard deviation). Since all values were well within the range of historical background data no toxicological significance was attached to this finding.

NEUROBEHAVIOUR
The variation in motor activity did not indicate a relation with treatment. No abnormalities were observed in any of the other functional observation tests. In two females of the 75 mg/kg/day dose group, animal No. 37 and No. 39, an increased and decreased motor activity was recorded, respectively, when compared to the activity recorded for the other animals in the study. Based on the absence of any corroborative findings in these animals and of a tendency within the dose group, no toxicological significance was attached to these findings.

ORGAN WEIGHTS
Organ weights and organ weights:body weight ratios of treated animals were considered to be similar to those of control animals.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The macroscopic findings noted were considered to be spontaneous in nature and within the range of biological variation for rats of this age and strain. The distribution of the findings did not distinguish treated animals from the controls.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Body weight gain

Target system / organ toxicity

Critical effects observed:

not specified