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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:
key study
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)
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[[5-acetamide-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-ethoxyphenyl]imino]diethyl diacetate
EC Number:
EC Name:
2,2'-[[5-acetamide-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-ethoxyphenyl]imino]diethyl diacetate
Cas Number:
Molecular formula:
2,2'-[[5-acetamide-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-ethoxyphenyl]imino]diethyl diacetate
Test material form:
solid: particulate/powder
Details on test material:
Stability: pure, stable. In vehicle stable minimum 2 hours
Storage: room temperature in the dark

Test animals

Details on test animals or test system and environmental conditions:
Mice are recommended for micronucleus assay as international standard.
Source: Kleintierfarm Madoerin AG, Switzerland
Age at the beginning of the test: 6 weeks (m), 7 weeks (f)
Initial bodyweight: 26-36 g (m), 23-34 g (f)
number of animals: 54 males and 54 females
Acclimation: 7 days at the test conditions under veterinary examination
Animals were randominzed
Method of identification: cages were labeled with project code, sex and dose. Animal identification by indelible inert color spots on the tail
Housing: groups of six animals
Cage: Makrolon Type 3 with wire mesh top and granulated softwood bedding
Environment: air conditioned temperature 22 ± 2 °C
Relative humidity: 55 ± 10 %
12 hours light/dark per day
Feed: pelleted standard Kliba 343-A, mouse diet ad libitum
Drinking: tap water ad libitum
and granulated :oftr¡ood bedding

Administration / exposure

Route of administration:
oral: gavage
2% carboxymethylcellulose and distilled water (suspension prepated just before the application). Homogeneity of the suspension was mantained during application by magnetic stirrer
Details on exposure:
The maximum tolerated dose was based on acute oral toxicity data. A preliminary acute oral LD50 (limit tes, two doses, 3 males and 3 females per dose) performed with the same mouse species as was used in this study showed the following results after 14 days of observation:
1000 mg/kg bw : 0 mortality in 6 animals
5000 mg/kg bw: 0 mortality in 6 animals
The 5000 mg/kg bw was used in this study as maximum tolerated dose.

the negative control received the test article vehicle, i.e. 2 % CMC in distilled water
the test group received 5000 mg/kg bw of test article (volume applied 20 ml/kg)
the positive control group received 50 mg/kg bw of cyclophosphamide (reference mutagen) dissolved in 0.9 % saline solution immediately before application

At 24, 48 and 72h after treatment, six mice per sex and group were sacrificed for examination. The first five animals of each sex were evaluated. The remaining animal of each sex was evaluated if macroscopic examination of the slides revelaed technical imperfections that may have prevented accurate microscopic analysis or if a test animal died even spontaneously or from gavage error.
Frequency of treatment:
Post exposure period:
72 h
Doses / concentrations
Doses / Concentrations:
5000 mg/kg bw
actual ingested
No. of animals per sex per dose:
18 males + 18 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):


Details of tissue and slide preparation:
AII mice were sacrificed by cervical dislocation. The femora were removed from each rmouse 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 Iabeled centrifuge tube.
The bone marrow celIs were dispersed in the calf serum as a homogeneous suspension. The tube containing the bone marrow ceIIs of both femora was centrifuged at 1000 r.p.m. fon 5 minutes.
The supernatant was removed, Ieaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette. Asmall drop of the marrow serum suspension was smeared on the sIide, 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.
The slides were coded before microscopic analysis. If rnacrocopic 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 nonmochromatic erythnocytes for micronuclei.
The calculated ratio polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 erythrocytes scored per sIide, measured the toxic efficacy of the test article.
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 Iikelihood method.
If a test article produced no statistically significant and reproducible positive response at any one of the test points, it was considered non-mutagenic in this system.

Results and discussion

Test results
Key result
no significant test article related increase of micronucleated polychromatic erythrocytes
no effects
Additional information on results:
No deaths occured. Sedation was observed in all test artcile treated animals for at least 6 hours after application.

Applicant's summary and conclusion

Under the study conditions, the test substance was not found to be genotoxic.
Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the test substance (93% purity) according to OECD Guideline 474. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Male and female rats (15/sex/group) were exposed to the test substance at concentration of 0 or 5000 mg/kg bw by gavage in a single application of a 2% carboxymethylcellulose (CMC) distilled water suspension. A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. The examinations were performed at 24, 48 and 72 h by sacrifice of 6 animals per sex. The substance did not show an increase of micronuclei from bone marrow compared to the vehicle control. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance was not found to be genotoxic (Archroma, 1985).