2,2'-[[5-acetamido-4-[(2-chloro-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

other: micronucleus assay - chromosome aberration

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[[5-acetamido-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

EC Number:

222-813-1

EC Name:

2,2'-[[5-acetamido-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

Cas Number:

3618-72-2

Molecular formula:

C23H25BrN6O10

IUPAC Name:

2-{[2-(acetyloxy)ethyl]({4-[ (E)-2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5acetamido-2-methoxyphenyl})amino}ethyl acetate

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

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

Vehicle:

2% carboxymethylcellulose and distilled water (suspension prepated just before the application). Hom
ogeneity 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:

once

Post exposure period:

72h

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

18 males + 18 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Examinations

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.
mperfections, 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 dist
ribution 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

Sex:

male/female

Genotoxicity:

negative

Remarks:

no significant test article related increase of micronucleated polychromatic erythrocytes

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

not measured/tested

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

Conclusions:

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).