Reaction products of diazotised 4,4-diaminodiphenylamine-2-sulfonic acid, subsequently coupled with 6-amino-4-hydroxynaphthalene-2-sulfonic acid, further diazotised and coupled with metaphenylendiamine, sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

other: esperimental data on similar substance

Adequacy of study:

key study

Study period:

From 17 February 1993 to 7 April 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Regarding the hypothesis for analogue approach, refer to the main endpoint summary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guidelines for testing of chemicals, section 4: Health Effects, 12.02.1981

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Directive 84/449/EEC of 25 04th 1984, published on the Official Journal of the European Communities L 251, 19 09th 1984, pp. 137

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Chemicals Act in the version published on 14 03. 1990 (Federal Law Gazette I. No. 13, p 521)

GLP compliance:

yes (incl. QA statement)

Remarks:

And standard operating procedures in their current version of the PFG BIOPHARM GmbH Berlin

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

EC Number:

229-208-1

EC Name:

Disodium 4-amino-3,6-bis[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxynaphthalene-2,7-disulphonate

Cas Number:

6428-31-5

Molecular formula:

C34H29N13O7S2.2Na

IUPAC Name:

disodium 4-amino-3,6-bis({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxynaphthalene-2,7-disulfonate

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:

TEST ANIMALS
- Source: Fa. Møllegaard, DE. Certificated
- Age at study initiation: from 6 to 8 weeks
- Weight at study initiation:
male: 27-48 g
female: 23-37 g
- Assigned to test groups randomly: yes, by PC-Program "RANDOM 2.1"
- Housing: box of Macrolon Typ 2. 5 animals per cage, males and female separated
- Bedding: Granular, Fa. Altromin food
- Diet: Standard diet for rat/mouse Altromin N 1326
- Water: ad libitum, tap water. In Glass bottle of 500 ml, with stainless steel lip
- Clinical control: three times at week during the acclimatation period and immediately after the administration of the test substance.
Animals were weighed randomly before the administration of the test substance

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 55 ± 10%
- Air changes: 10 times; automatic system Geringfugugige Abweichungen
- Photoperiod: automatic regulation of 12h/12h (light period from 6:00 a.m to 6:00 pm)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.7% aqueous amylopectin Ultra with the addition of 2 drops of Tween 80 per 10 ml of preparation

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Pre-test: To determine the maximum tolerated acute dose. LD50 at 48 h was > 1600 mg/Kg, test substance at 80%. Following a single administration animals were observed over a period of 48h. Since none of the treated animals died, this dose was used for the main test.
- Main test: the maximum dose was decreased to 1200 mg/ Kg, because a dose of 1600 mg/kg given to 2 groups of males animals produced some effects. At 800 and 400 mg/Kg no effects were observed on male and female mice.

The substance volume of application has been determined on the basis of body-weight (0.2 ml/10 g bw).

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Single oral administration for the tested item

Post exposure period:

At 24 and 48 h

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1600, 1200, 800, 400 kg/kg
Basis:
nominal in diet
Males

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1600, 800, 400 kg/kg
Basis:
nominal in diet
females

No. of animals per sex per dose:

85 mouse (50 male and 35 female), 5 animals for sex and group

Control animals:

yes

Positive control(s):

The preparations of the positive control substance were freshly prepared before administration.
- Substance: Cyclophosphamide
- Source: SIGMA CHEMIE GmbH
- Lot: 19F-0254
- Route of administration: intraperitoneal injection
- Doses / concentrations: 80 mg/kg (0,1 ml/10g bw in aqua)
- Solvent: water for injection

The positive and negative control substances were processing after 24 hours.

Examinations

Tissues and cell types examined:

For each animal were examined microscopically 2000 polychromatic erithrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The initial dose of 1600 mg/kg was used, up 80% of the client specified LD50 value of the test substance. After a single oral administration of the initial dose, the animals were observed for the occurrence of intoxication and mortality over a period of 48 hours. Since none of the treated animals had died, this dose was used for the main test. In the main test, however, in the male animal collective group 1 and 11, the tested item in the dose of 1600 mg/kg cause the dead within 24 hours of the animals. The maximum tolerated dose was lowered to 1200 mg/kg and used in the test. The other doses of 800 and 400 mg/kg proved to be non-toxic and have been tested in male and female animals, as provided in the main test.

OBSERVATION
After administration of the maximum tolerated dose, the bone marrow was analysed for 24 and 48 h; for the other two doses the analysis of the bone marrow was performed after 24 hours.

DETAILS OF SLIDE PREPARATION
Animals were killed by cervical dislocation. Removal of the femurs, extraction of the bone marrow and production of slide preparations. The femurs were dissected immediately after killing and removed. 1 ml of foetal calf serum has been warmed to 37°C in a water batch and measured in a syringe of 2ml. The open needle was inserted into the open end of the femur. The marrow has been flushed out.
The resulting cell suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed using a Pasteur pipette; 4 drops of foetal calf serum Pipette were added to the sediment and resuspended the cells in it.
The cells suspension was treated and the specimens coloured following Pappenheimer's method.
3 slides per animal were prepared.

METHOD OF ANALYSIS
2000 polychromatic erythrocytes per animal were examined microscopically in order to determine the presence and the number o micronuclei.

Evaluation criteria:

The classification of micronuclei was performed using the following criteria:
- Micronuclei are round, rarely oval or crescent shaped.
- They have a sharp contour.
- They are uniformly coloured and are approximately from 1/20 to 1/5 of the diameter of the erythrocytes.
- It is usually present only a micronucleus.
- After treatment with high doses of substances that produce chromosome breaks, in some erythrocytes can also occur several micronuclei, which can also be almond shaped or annular.

The ratio of polychromatic to normochromatic erythrocytes was determined individually for each animal by counting a total of 1000 Erylhrczyten.

Statistics:

The evaluation of micronuclei, the ratio of polychromatic to normochromatic erythrocytes in the experimental groups and comparison groups to which the substance was administered with control groups have been prepared in accordance with the "Statistical analysis of data for micronucleus test".
Polychromatic erythrocytes with micronuclei data (PCE's), total number of micronuclei (MN gas) and ratio of normochromatic to polychromatic erythrocytes (PCE's to NCE's)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Only 2 males died at dose level of 1600 mg/kg

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Clinical signs of toxicity in test animals: no signs in females at 1600, 800 and 400 mg/kg; 2/10 males administrated with a single dose of 1600 mg/kg died

Maximum tolerable dose:
- Males: 1200 mg/kg
- females: 1600 mg/kg

At 1600 mg/kg: no systematic effects observed in females; two dead in males.
At 1200 mg/kg: no systematic effect in males
At 800 and 400 mg/kg: no symptoms for both males and females.

Any other information on results incl. tables

POLYCHROMATIC ERYTHROCYTES

The analysis of polychromatic Erythrocytes in the presence of micronuclei and the subsequent statistical evaluation, justify the following statement:

under the test conditions described, no increased incidence of micronucleated polychromatic containing Erythrozyien from mouse bone marrow was observed. Thus, a potential can be excluded for the induction of chromosomal damage or damage ending of the mitotic apparatus by the test substance in this in vivo short-term test.

In both male and female animals and in any of the doses tested, an increased occurrence of micronuclei in polychromatic erythrocytes from mouse bone marrow was observed.

The calculated frequency of micronucleated polychromatic erythrocytes in groups treated with test substance was 0.17 to 0.42% and thus had no differences in the values ​​of the negative controls at 0.19 to 0.28%.

In almost all animal has been noted a slight increase in the ratio of PCE:NCE (range of 1:1.20 to 1:1.56)

CONTROLS RESULTS

The positive control substance cyclophosphamide resulted in an incidence of 1.78% with a significantly increased incidence of micronucleated PCE-containing. Chance was more than a micronucleus contained in a PCE, which is attributed to the strong mutagenic potential of this substance [2].

Regarding the relationship between polychromatic and normochromatic erythrocytes and the corresponding negative controls, no differences were observed.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Potential for the induction of chromosomal damage or damage ending of the mitotic apparatus by the test substance can be excluded.

Executive summary:

Gene mutation micronucleus assay was performed according to a recognized method: Directive 84/449/EEC of 25 04th 1984, published on the Official Journal of the European Communities L 251, 19 09th 1984, pp. 137.

Substance was administrated by oral gavage to mouse at dose concentrations of 1600, 1200, 800, 400 kg/kg in male and 1600, 800, 400 kg/kg in female.

Conclusion

Potential for the induction of chromosomal damage or damage ending of the mitotic apparatus by the test substance can be excluded.