2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

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:

Oct 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

EC Number:

404-910-2

EC Name:

2-(4-(diethylaminopropylcarbamoyl)phenylazo)-3-oxo-N-(2,3-dihydro-2-oxobenzimidazol-5-yl)butyramide

Cas Number:

164578-14-7

Molecular formula:

C25H31N7O4

IUPAC Name:

N-[3-(diethylamino)propyl]-4-[(1E)-2-{2-oxo-1-[(2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)carbamoyl]propyl}diazen-1-yl]benzamide

Test material form:

solid: bulk

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation:
- males: 29.6 g (23-34 g)
- females: 23.8 g (21-27 g)
- Housing: in fully air-conditioned rooms in Macrolon cages type 3, on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): rat/mice diet Altromin 1324 (Altromin GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%): 55+/-10%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 16. Oct. To: 19. Oct. 1989

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil
- Justification for choice of solvent/vehicle: homogenity of test item in suspension
- Concentration of test material in vehicle: 25% (w/v)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw diveded into two equal parts of 10 mL/kg bw, administered within 2 h

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test compound dilutions were prepared fresh each day. 6250 mg of test substance were weight in a beaker, mixed with sesmae oil, washed out in a 25 mL flasked and topped up to the calibration mark. A suspension was formed.

Frequency of treatment:

two equal portions within 2 h

Post exposure period:

animals were killed 24, 48 or 72 h after application (sampling of bone marrow)

Doses / concentrations

Dose / conc.:

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

Remarks:

based on preliminary study for acute toxicity

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan (cyclophosphamid)
- Justification for choice of positive control(s):
- Route of administration: gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

immature erythrocytes of the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on preliminary test for acute toxicity

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
one oral application (gavage; two equal volumes within 2 h)
sampling times: 24, 48 and 72 h adfter application

DETAILS OF SLIDE PREPARATION:
For each animal approx. 3 mL FCS was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 min at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was semared on a cleaned slide, and air-dried for approx. 24 h.
Staining procedure.
5 min methanol, 3 min May-Grünwalds solution, 2 min May-Grünwalds solution diluted 1:1 with distilled water, rinsing with dstilled water, 10 min staining with Giemsa, rinsing with distilled water, coating with Entellan

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted ans examined for micronuclei. In addtition, the ratio of polychromatic to normochromatic erythrocytes was determined.

Evaluation criteria:

according to OECD guideline 474, 1983

Statistics:

according to Wilcoxon (paired, one-sided increase)

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after application and showed no signs of toxicity.
The incidence of micronucleated polychromatic erythrocytes in the dose dose groups of the test item was within the normal range of the negative control groups. No statistically significant increase of micronucleated plychromatic erythrocytes has been observed. The number of normchromatic erythrocytes with micronulcei did not differ significantly from the values of the simulatneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test comound.
Cyclophosphamid induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity to the test system.

Applicant's summary and conclusion

Conclusions:

The results of this in vivo MNT in mice indicate that, under the conditions of the present study, the test substance is not mutagnic in the micronucleus test.

Executive summary:

The test item was tested in the in vivo MNT. It was administered orally by gavage to male and female mice in doses of 0 and 5000 mg/kg bw.

The high dose was chosen since a preliminary study had shown it to be the maximum appicable dose.

The test compound was given in two equal parts within two hours and according to the test procedure the animals were killed 24, 48 or 72 h after administration. Cyclophosphamid was used as positive control substance and administered orally at a dose of 50 mg/kg bw. The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item was within the normal range of the negative control. The number of normochrmatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was statistically not different from the control values. Cyclophosphamid induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes showed a difference to the negative control values.