Imidazole

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with OECD test guideline 474 for Micronucleus test and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Imidazole

EC Number:

206-019-2

EC Name:

Imidazole

Cas Number:

288-32-4

Molecular formula:

C3H4N2

IUPAC Name:

1H-imidazole

Details on test material:

Imidazole as hydrochloride, degree of purity 99.5%

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Weight at study initiation: mean weight of about 28g

HOUSING
For the duration of about one week the animals were housed in Makrolon cages, type M III, in groups of 5 separately according to sex in fully air-conditioned rooms in which central air conditioning guaranteed a range of 20 - 24°C for temperature and a range of 30 - 70% for relative humidity . Before the start of the treatment the animals were transferred to Makrolon cages, type M I, and housed individually under the same conditions until the end of the test.

- Diet (ad libitum): standardized pelleted feed (Kliba Haltungsdiät, Klingentalmühle AG, Kaiseraugst, Switzerland)
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Vehicle used: olive oil; dose volume of 10 ml/kg bw. Concentrations were 5, 10,and 20 g/100 ml oil.
Substance stability was analytically verified.

Duration of treatment / exposure:

Imidazole was singly administered by oral gavage in olive oil.

Frequency of treatment:

One treatment

Post exposure period:

Test item group animals dosed at 2000 mg/kg bw were sacrificed 16, 24 and 48 hrs following dosing.
Test item group animals dosed at 500 or 1000 mg/kg bw were sacrificed 24 hrs following dosing.
Vehicle control animals were sacrificed 24 hrs following dosing.
Positive control animals were sacrificed 24 hrs following dosing.

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw (suspended in 10 ml olive oil/kg bw)
Basis:
nominal conc.

No. of animals per sex per dose:

70 NMRI mice, male and female, mean bw 28 g. 5 animals per dose and sex were used for TS and negative control, and 5 animals
(2 and 3 per sex) for each of the positive control substances.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive controls received once orally the clastogen cyclophosphamide (20 mg/kg bw, in water, gavage at 10 ml/kg bw, 2 males/ 3 females) or the spindle poison vincristine (0.15 mg/kg bw, in water, i.p. at 10 ml/kg bw, 3 males/ 2 females).

Examinations

Tissues and cell types examined:

The animals were examined for clinical signs of toxicity after dosing. Femural bone marrow was prepared from all control and dose group
animals at 24 hr post dosing, and additionally at 16 hr and 48 hr in animals receiving the high 2000 mg/kg bw dose.

Details of tissue and slide preparation:

Preparation of the bone marrow
The two femora were prepared from the animals, and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with
ground edges, the preparations were dried in the air and subsequently stained.

Staining:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest . and clarified in xylene,
the preparations were embedded in Corbit-Balsam.

Slides were coded before microscopic analysis.

Evaluation criteria:

Generally, 1000 polychromatic erythrocytes per animal are evaluated for the parameters:
(1) no. of polychromatic erythrocytes with/without micronuclei and calculation of clastogenic index,
(2) no. of normochromatic erythrocytes with/without micronuclei, calculation of ratio polychromatic/normochromatic erythrocytes,
(3) no. of small(d<1/4) and large (d>1/4) micronuclei.
(d= diameter of micronucleus, D= cell diameter)

Statistics:

No statistical data analysis was necessary to be performed. The number of polychromatic micronucleated erythrocytes after TS treatment was nearly the range of the actual control value, and within the historical values.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs of sytemic toxicity were present at all dose levels

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
During pretests deaths were observed down to a dose of 2250 mg/kg bw whereas 2000 mg/kg bw was survived by all animals but led
to signs of toxicity including irregular respiration, piloerection, squatting posture in some cases, closed eyelids, and saltatory convulsions.

RESULTS OF DEFINITIVE STUDY
Analytical examination of olive oil samples revealed concentrations of 52.9, 100.4, and 198.0 g/l, i.e. 99-106% of the theoretical values.

- Induction of micronuclei:
The single oral administration of olive oil in a volume of 10 ml/kg bw led to 2.6% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.
After the single administration of the highest dose of 2000 mg/kg bw, 1.9% polychromatic erythrocytes containing micronuclei were found
after 16 hours, 2.3% after 24 hours and 1.4 % after 48 hours.
In the two lower dose groups rates of micronuclei of about 1.7% (1000 mg/kg bw group) and 2.2% (500 mg/kg bw group) were detected
after a sacrifice interval of 24 hours in each case.
With 8.6 % the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg bw.
With 100% the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing
polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 15.4%.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.
Thus, the test substance imidazole-hydrochlorid did not lead to any increase in the rate of micronuclei.
The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any of the sacrifice intervals.

- Ratio of PCE/NCE:
The ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the
control values in all dose groups.

- Clinical signs:
The single oral administration of the carrier in a volume of 10 ml/kg bw was tolerated by all animals without any signs or
symptoms. Doses of 2000 mg/kg bw and 1000 mg/kg bw led to irregular respiration, piloerection and in few cases to closed eyelids about 15 - 30 minutes after treatment of the animals; in the 2000 mg/kg group in some animals squatting posture was additionally observed and one animal was found dead 1 hour after test substance administration. In the 500 mg/kg bw group only irregular respiration and piloerection were found about 15 - 30 minutes after test substance treatment. The day after treatment clinical signs were not observed in all test groups anylonger. Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg bw nor that of vincristine in a dose of 0.15 mg/kg bw caused any evident signs of toxicity.

- Statistical evaluation:
No biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent
control and the 3 dose groups (2000 mg/kg, 1000 mg/kg and 500 mg/kg) or between the various sacrifice intervals (16, 24 and 48 hours)
was observed.

Applicant's summary and conclusion

Executive summary:

Imidazole hydrochloride was tested in a micronucleus test in accordance with the OECD TG 474 under GLP conditions in mice, dosed by gavage with 500, 1000, and 2000 mg/kg bw. The salt imidazole hydrochloride dissociates into protonated imidazole and chloride in the stomach following oral gavage and did not induce micronuclei at any dose or any harvesting time, which were set at 16, 24, and 48 hours first after dosing. The animals showed signs of toxicity at 500 mg/kg bw and above. The number of polychromatic and normochromatic erythrocytes was not statistical significantly different from the control, therefore it may be concluded, that imidazole was not toxic to the bone marrow. Imidazole was found to be not clastogenic or aneugenic in this test.