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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-11-21 to 1983-12-21
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
26 May 1983 (first addendum)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
C14 H6 Cl2 F4 N2 O2
Test material form:
solid: crystalline

Test animals

KFM (outbred, SPF-quality)
Details on species / strain selection:
Mice are recommended for micronucleus assays as the international standard.
Details on test animals or test system and environmental conditions:
- Source: Kleintierfarm Madoerin AG, 4414 Fuellinsdorf / Schwitzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: 22-41 g
- Assigned to test groups randomly: Animals were randomized into the different groups after delivery using a random algorithm.
- Fasting period before study: no
- Housing: groups of 6, Makrolon Type 3, with wire mesh top
- Diet: ad libitum, pelleted standard Kliba 343-A mouse diet
- Water: ad libitum, tap water
- Acclimation period: 7 days

- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): no data, air-conditioned room
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 250 mg/mL
- Amount of vehicle: 20 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: A suspension was prepared immediately before application by adding the test material to distilled water containing 2% vehicle CMC. The suspension was then homogenized. Homogeneity of the suspension was maintained during application using a magnetic stirrer. Dosing suspensions prepared immediately prior to administrationwere stable for at least 2 hours.
Duration of treatment / exposure:
24, 48 and 72 hours
Frequency of treatment:
Post exposure period:
24, 48, 72 hours
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw (total dose)
No. of animals per sex per dose:
6 (only 5 animals were used for analysis)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Justification for choice of positive control: accepted reference mutagen
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw dissolved in 0.9% saline


Tissues and cell types examined:
All mice were sacrificed by cervical dislocation. The femora were removed from each mouse and freed of adherent tissue. The proximal end of the femur was cut with scissors. The needle of a plastic syringe containing 0.2 mL calf serum was inserted into the proximal part of the marrow canal which was closed at the distal end. The femur was submerged in 1.5 mL calf serum in a labeled centrifuge tube. The bone marrow cells were dispersed in the calf serum as a homogeneous suspension. The tube containing the bone marrow cells of both femora was centrifuged at 1000 r.p.m. for 5 minutes. The supernatant was removed, leaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose applied was based on acute oral toxicity data. The acute oral LD50 study with the mice species used in this experiment indicated a LD50 value greater than 5000 mg/kg body weight (see Section 7.2.1). The dose of 5000 mg/kg body weight was used in this experiment as the expected maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: At 24-, 48- and 72-hour intervals after treatment, 6 mice per sex and group were sacrificed for examination. The first 5 animals of each sex were evaluated.

DETAILS OF SLIDE PREPARATION: A small drop of the marrow serum suspension was smeared on the slide, which was identified by project code and the animal number, and allowed to dry overnight. Two slides per animal were prepared. The following day, the smears were stained using the panoptic stain method developed by Pappenheim .

METHOD OF ANALYSIS: The slides were coded before microscopic analysis. If macroscopic evaluation revealed technical imperfections, the first slide was replaced by the second slide prepared. From each animal, one thousand polychromatic erythrocytes (PCE) were scored under the microscope (magnification 1000x), for the incidence of micronuclei. Additional information could be obtained by scoring normochromatlc erythrocytes for micronuclei. The calculated ratio polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 erythrocytes scored per slide, measured the toxic efficacy of the test material.

Evaluation criteria:
The frequencies of micronuclei of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution was applied. Estimation and testing were performed by maximum likelihood method. A test material which produced no statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
Poisson heterogenicity test, Maximum Likelihood Method

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
After a single application of the test material at 5000 mg/kg body weight by gavage, no significant substance-related increase of micronucleated polychromatic erythrocytes was observed in either male and female treated groups when compared with corresponding negative control groups. These results were found in the three examination times, 24, 48 and 72 hours post-application, respectively. No evidence of toxic effects of the test material was observed. The positive control groups receiving cyclophosphamide exhibited a significant and clear increase in the number of micronucleated polychromatic erythrocytes and thus validated the test. The dose used for this reference mutagen exhibited a toxic effect as shown by the reduced PCE/NCE ratio.
- Appropriateness of dose levels and route: No treatment-related symptoms and no death occurred during the application period in this study. From ADME studies in rodents (see section 7.1) systemic exposure after oral gavage was reported, however absorption of the substance in rodents was reported to be low and independent of the dose level and sex. Following administration of a single high dose of 750 mg/kg bw in male rats nearly constant plasma levels were reached during the first 8 hours post treatment (1.17 -1.72 µg/mL). A peak mean concentration of 3.27 µg/mL occurred at 24 hour in males. In females, the corresponding values were 0.98 -1.43 µg/mL. From the 2nd day post-treatment, the plasma levels declined to reach 0.06 and 0.08 µg/mL after 7 days in males and females respectively. From all available ADME studies an absorption rate of <10% of the dose can be estimated. However, exposure of the target tissue in this in vivo micronucleus test is expected at least for the 24 h time point. In an acute oral toxicity study in which the same mouse stain and batch of the test item have been used the LD50 was > 5000 mg/kg bw and dyspnea and ruffled fur were observed in all mice as clinical symptoms indicating systemic exposure (see section 7.2).
- Statistical evaluation: There was no statistical evidence for differences between the negative control groups and the corresponding treatment groups.

Any other information on results incl. tables

Mean micronuclei scored at different time points following treatment with the test substance:

Test group

24 hours post-application

48 hours post-application

72 hours post-application


Mean # micronuclei ± SD

Mean Ratio PCE/NCE


Mean # micronuclei ± SD

Mean Ratio PCE/NCE


Mean # micronuclei ± SD

Mean Ratio PCE/NCE

Vehicle control

(2% CMC)


1.1 ± 0.74



0.3 ± 0.48



0.6 ± 0.70


Test group

(5000 mg/kg bw tet substance)


1.5 ± 0.97



1.1 ± 0.88



0.8 ± 0.92


Positive control

(50 mg/kg bw CPA)


32.9 ± 5.99



20.9 ± 4.90



7.7 ± 1.42



Applicant's summary and conclusion