Sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(3-(4-amino-9,10-dihydro-3-sulpho-9,10-dioxoanthracen-4-yl)aminobenzenesulphonyl)vinyl) disodium sulphate

EC Number:

219-949-9

EC Name:

2-(3-(4-amino-9,10-dihydro-3-sulpho-9,10-dioxoanthracen-4-yl)aminobenzenesulphonyl)vinyl) disodium sulphate

Cas Number:

2580-78-1

Molecular formula:

C22H18N2O11S3.2Na C22H18N2Na2O11S3

IUPAC Name:

disodium 1-amino-9,10-dioxo-4-[(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Blue 19

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males mean= 30.5 g (26 - 35 g); females: mean= 25.6 g (23 - 31 g)
Acclimatization: at least 5 days
Food / water: mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum; tap water in plastic bottles, ad libitum
Housing: 5/cage
Room temperature: 22 +/- 2°C
Relative humidity: 55 +/- 10%
Lighting time: 12 h/12 h light/dark cyclus

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionised water

Details on exposure:

The dose levels for micronucleus testing were selected on the basis of a preliminary study to determine the acute toxicity and the maximal applicable dose. Oral administration of 5000 mg/kg bodyweight did not lead to a partial lethality in male and female mice. It is considered the maximal applicable dose and was selected as dose level for the main study.

The 5000 mg/kg bw dose was prepared as a 25% (w/v) solution and administerd at a volume of 20 mL/kg bw devided into two equal parts of 10 mL/kg bw administered within 2 hours

The test compound dilutions were prepared fresh each day. 6250 mg test substance were weight in a beaker, mixed with deionisized water, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

Duration of treatment / exposure:

animals were killed after 24, 48 or 72 hours after administration of the test compound

Frequency of treatment:

The test compound was given in two equal parts within two hours

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 for each killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

endoxane, 50 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum 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 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Staining procedure
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- 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:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison
of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).

Statistics:

The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the I1Diamant l1 computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 %level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All animals survived after application of 5000 mg/kg bodyweight. The following signs of toxicity were observed: faeces dark blue coloured and diarrhea. 24 hours p.a. in the cages no. 3, 4, 9 and 14 the soft-wood granulate was red coloured.
48 hours after application all animals were free of clinical signs of toxicity.
The dissection of the animals revealed the following macroscopic findings: At the killing time 24 hours p.a. at the dosage 5000 mg Remazol-Brillantblau R FWTRG per kg bw in some male and females the content of appendix and colon was blue coloured.

The incidence of micronucleated polychromatic erythrocytes in the treated groups was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Applicant's summary and conclusion

Conclusions:

No clastogenic or aneugenic effects were noted

Executive summary:

The test substance was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg per kg bodyweight.

The 5000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

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 hours after administration of the test compound.

EndoxanR was used as positiv control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The number of normochromatic 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.

Endoxan(R) 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 was not changed to a significant extend.

The results indicate that, under the conditions of the present study, the test substance is not mutagenic in the micronucleus test.