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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1987-10-06 to 1987-10-13
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
as described in the Annex of EEC Directive 87/449
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
(±)-1-(β-allyloxy-2,4-dichloro-phenylethyl) imidazole / (±)-allyl 1-(2,4-dichlorophenyl)-2-imidazol-1-ylethyl ether
Test material form:
solid: crystalline
Details on test material:
- Physical state: crystalline solid
- Appearance: Yellow to brown crystalline solid
Specific details on test material used for the study:
- Source and lot/batch No.of test material: ZR 23979 D 8801
- Expiration date of the lot/batch: not specified
- Purity test date: 1987-12-03
- manufacturing date: 1987-06-17

- Storage condition of test material: at room temperature in closed containers
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test article is determined before the initiation of the study and a reserve sample from each batch of the test article is retained for a period of minimum 5 years in the analytical department.

FORM AS APPLIED IN THE TEST: suspended in propylene glycol

Test animals

Details on species / strain selection:
The species and strain is selected following review of available data on toxicity, pharmacology and/or pharmacokinetics and also because of the possibility to compare the recorded data with the historical controls of the laboratory.
Details on test animals or test system and environmental conditions:
- Source: Albino Swiss mice, supplied by own permanent non-inbred laboratory colony (established in 1956)
- Age at study initiation: 5 weeks
- Weight at study initiation: Males – 33 to 35 g ; Females: 31 to 35 g
- Assigned to test groups randomly: not indicated
- Fasting period before study: not indicated
- Housing: individually housed in a numbered macrolon cage
- Diet (e.g. ad libitum): "Huybrechts" pelleted diet, administered in self-raising hoppers, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least one week

- Temperature (°C): 20 ± 2 °C
- Humidity (%): 60 ± 10%
- Air changes (per hr): approximately 18 per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: unspecified
- Vehicle(s)/solvent(s) used: Polyethylene glycol
- Justification for choice of solvent/vehicle: not indicated
- Concentration of test material in vehicle: not specified
- Amount of vehicle: 0.1 mL per 10 g bw
Details on exposure:
The solutions are only freshly prepared formulations. All animals received a single standard volume of 10 mL/kg body weight orally.
Duration of treatment / exposure:
one single administration
Frequency of treatment:
one single administration
Post exposure period:
And sampling of the bone marrow was done 24, 48 and 72 hours after dosing
Doses / concentrationsopen allclose all
Dose / conc.:
20 other: mg/kg bw
low dose; 24h, 48h and 72h sacrifice time
Dose / conc.:
80 other: mg/kg bw
medium dose; 24h, 48h and 72h sacrifice time
Dose / conc.:
320 other: mg/kg bw
high dose; 24h, 48h and 72h sacrifice time
No. of animals per sex per dose:
The 130 animals are divided into 13 groups, each of 5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide, sacrifice time 48h only.
Justification for choice of positive control(s): no
Route of administration: orally, once
Doses/ concentrations: dissolved in 0.4M Tartaric Acid + water, dose 40 mg/kg bw
As only freshly prepared formulations of the test article are used no stablilty of the preparations is required.


Tissues and cell types examined:
Bone marrow: polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In order to determine the maximum tolerable dose level for the main micronucleus test, a preliminary toxicity test is performed with three or four dosage groups of 3 male and 3 female rats or mice.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. One femur per animal is dissected free and the bone marrow is rinsed out with fetal calf serum. After centrifugation a drop of the homogenous cell suspension is placed on a slide and spread as a smear with a blood smearing instrument. Slides are then coded to avoid subjective bias. Slides are left to dry and are fixed with methanol and stained with Wright on an automatic apparatus. Finally, the slides are permanently mounted.

METHOD OF ANALYSIS: The slides are examined by light microscopy (magnification 1000x). Aa total of one thousand polychromatic erythrocytes (PCE) are counted per animal and the number of micronucleated polychromatic erythrocytes are recorded. At the same time, the number of micronucleated normochromatic erythrocytes (NCE) are also recorded in the fields containing these 1000 polychromatic erythrocytes.
The ratio of polychromatic erythrocytes to normochromatic plus polychromatic erythrocytes is determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
A test chemical is considered positive in the micronucleus test if it induces a statistically significant (p < 0.05) dose-related increase in the number of micronucleated polychromatic erythrocytes in the combined data for both sexes or in the data for male or female groups separately.

A test chemical is considered negative in the micronucleus test if none of the tested concentrations or sampling times showed a statistically significant (p < 0.05) increase in the number of micronucleated polychromatic erythrocytes either in the combined data for both sexes or in hte data for male or female groups separately.
The preceding cirteria are not absolute and other extenuating factors may enter into the final evaluation decision.
The significance of any inter-group differences is assessed by the Mann-Whitney U test.

Results and discussion

Test results
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
- Dose range: 20 mg/kg bw; 80 mg/kg bw; 320 mg/kg bw
- Solubility: not reported
- Clinical signs of toxicity in test animals: In a preliminary toxicity study, the test item was administered once orraly to three male and three female mice at dose levels of 160, 320 and 640 mg/kg. All the males and females of the 640 mg test item / kg dosage group died at 24h after dosing. One male of the 320 mg test item / kg dosage group died at 48h after dosing.
- Evidence of cytotoxicity in tissue analyzed: The number of polychromatic erytghrocytes (PCE) to 200 PCE and normochromatic erythrocytes (NCE) examined was reduced in the 160 and 320 mg test item / kg dosage groups. The body weight of the 160 and 320 mg test item / kg treated males and females was also found to be strongly reduced.
On basis of the dose related increase in mortality, reduction in bonne marrow proliferation and decrease in body weight found with the 160, 320 and 640 mg/kg treated mice, 320 mg/kg bw was considered as the highest acceptable dose level for the main test.

- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/NCE (for Micronucleus assay): no increase in the number of micronucleated PCE was detected at any of the sampling times when male and female mice were dosed once with 20, 80 and 320 mg test item / kg orally. This means that up to a toxic dose level the test item did not show chromosome breaking or spindle poisoning activity resulting in the formation of micronuclei.
- Clinical signs of toxicity in test animals: In the 20 and 80 mg test item / kg dosage groups, all the animals survived the experimental period. In the 320 mg test item / kg dosage group two females of the 24h sampling time, one male and three females of the 48h sampling time and three males and one female of the 72h sampling time, died before sacrifice time.
The body weight of the 320 mg test item / kg treated animals was found to be significantly decreased (p<=0.01) at the 48 and 72h sampling times when compared to the solvent control group. The bone marrow proliferation of the 320 mg test item treated mice was also found to be significantly decreased (p <= 0.01 - p<=0.001) at the 48 and 72h sampling time.

The cumulated laboratory background solvent control data on PCE of males and females show a mean of 1.1 micronucleated PCE / 1000 PCe with a range of 0-5/1000 PCE. At the sacrifice times, the rate of micronuclaeted PCE in the solvent control group falls within the laboratory background solvent control range .
The positive control shows at the 48h sacrifice time a significant increase (p<= 0.001) increase in the number of micronucleated PCE as a result of the chromosome breaking activity of cyclophosphamide.

Applicant's summary and conclusion

The test item did not show any potential for inducing micronuclei in the erythrocytic system in male and female mice.