N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

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:

weight of evidence

Study period:

From October 2002 to November 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

EC Number:

287-466-0

EC Name:

N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

Cas Number:

85508-41-4

Molecular formula:

C21H21ClN6O6

IUPAC Name:

N-{2-[(2-chloro-4,6-dinitrophenyl)diazenyl]-5-(diallylamino)-4-methoxyphenyl}acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Name in test report: Disperse Blue 291.1

Specific details on test material used for the study:

Stability and homogeneity in the vehicle: guaranteed for 4 hours in Tylose H4000 G4 PHA 0.5% by HPLC analysis

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchcn
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male animals-mean = 181.2 g (= 100 %) (min = 175 g (-3.4 %), max = 192 g (+6 %)); female animals- mean = 142.5 g (=100 %) (min = 137 g (-3.9 %), max = 150 g (+5.2 %))
- Assigned to test groups randomly: Yes
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-H (V 1534), ad libitum, ssniff® GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Tylose H4000 G4 PHA 0.5%
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SUSPENSION: The test substance was suspended in Tylose H4000 G4 PHA 0.5% at an appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

two doses separated by an interval of 24 hours

Frequency of treatment:

twice

Post exposure period:

24 hours

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5/sex/group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:

- 2,000 polychromatic erythrocytes were counted for each animal.
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2000 mg/kg bw was selected as the limit dose, for the main study.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.

-Staining procedure: The slides were stained as follows:-
-5 minutes in methanol
-5 minutes in May-Grunwald's solution
-brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-rinsing in distilled water
-drying
-coating with Entellan

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.

Statistics:

Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

red discoloured urine 5-6 hours after the administration prove exposure to the test substance

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment. No signs of toxicity were detected, but all dosed animals showed red discoloured urine 5-6 hours after the administration.
The dissection of the animals revealed dark blue coloured contents of the gastro-intestinal tract.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test substance was suspended in Tylose and given twice at an interval of 24 h as a oral dose of 2000 mg/kg bw/d to male and female rats. The dose was selected based on the results of a previous rat acute oral toxicity study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. After treatment with the test substance, the number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected and differed less than 20% from the control value.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.