2,7-Naphthalenedisulfonic acid, 4-amino-5-hydroxy-, coupled with 3-aminophenol, diazotized 5-amino-2-[(4-aminophenyl)amino]benzenesulfonic acid and diazotized benzenamine, sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January from 02 to 26, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26th, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Direct Black 168

IUPAC Name:

Direct Black 168

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH- 4414 Füllinsdorf/Basel, Switzerland.
- Age at start of acclimatization: minimum 10 weeks.
- Weight at study initiation: approx. 30 g.
- Assigned to test groups randomly: yes.
- Fasting period before study: yes, approximately 18 hours before treatment with the test article. Animals could still have water ad libitum during the fasting period.
- Housing: single in a Makrolon Type I, with wire mesh top and granulated soft wood bedding.
- Diet: pelleted standard diet.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C.
- Humidity: not regulated.
- Photoperiod: 12 hrs dark / 12 hrs artificial light (6.00 a.m - 6.00 p.m).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: destilled water.
- Justification for choice of vehicle: chocen for its non-toxicity to the animals.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test article was dissolved in distilled water and the animals received orally a single standard dose volume of 20 ml/kg b.w.

Frequency of treatment:

Once at the start of the study.

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

Three groups

No. of animals per sex per dose:

6 males and 6 females per dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- Preparation: the substance was dissolved in physiological saline and the solution was prepared on the day of administration.
- Route of administration: orally.
- Dosing: single dose of 40 mg/kg b.w.
- Volume administered: 10 ml/kg b.w.
- Stability: at 20 °C only 1 % of CPA is hydrolysed per day in aqueous solution.

Examinations

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

PREPARATION OF THE ANIMALS
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and
the supernatant was discarded.

DETAILS OF SLIDE PREPARATION
A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample

ANALYSIS OF CELLS
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

CRITERIA FOR DOSE SELECTION
A preliminary study on acute toxicity was performed with the same strain and under identical conditions as in the mutagenicity study. 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w of the substance dissolved in aqua dest. The volume administered was 20 ml/kg b.w.
The maximum tolerated dose or the highest dose that can be formulated and administered reproducibly sould be selected. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hours. Higher dosing than 5000 mg/kg b.w. was not attainable as appropriate solutions could be obtained only up to 250 mg/ml and application volumes higher than 20 ml/kg b.w. were not justifiable for the animals used.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

slight toxic reactions

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

POSITIVE CONTROL
40 mg/kg b.w. cyclophosphamide showed a distinct increase of induced micronuleus frequency.

PRE-EXPERIMENT FOR TOXICITY
All treated animals expressed slight toxic reactions such as reduction of spontaneous activity. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the substance had no cytotoxic properties.

Any other information on results incl. tables

The number of PCEs (Polychromatic Erythrocytes) with micronuclei and the ratio of PCE to NCE ( Normochromatic Erythrocyte) are given for each test group in the table below.

Summary of results.

Test group	dose (mg/kg bw)	sampling time (h)	PCEs with micronuclei	range	PCE/NCE
solvent	0	24	0.09 %	0-3	1000/935
test article	5000	24	0.09 %	0-3	1000/874
cyclophosphamide	40	24	0.76 %	3-11	1000/1119
solvent	0	48	0.02 %	0-2	1000/842
test article	5000	48	0.04 %	0-1	1000/1000
solvent	0	72	0.07 %	0-2	1000/892
test article	5000	72	0.03 %	0-2	1000/769

Applicant's summary and conclusion

Conclusions:

The test article did not induce micronuclei in the bone marrow cells of the mouse.

Executive summary:

The substance was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, according to the OECD Guideline 474 and EU Method B12. Six male and six female mouses were treated orally with a single dose of 5000 mg/kg b.w. This was the maximum attainable dose as estimated by the pre-experiment. Concurrent positive (use of cyclophosphamide) and negative (use of vehicle-distilled water) control groups run. After 24 h, 48 h and 72 hours the bone marrow cells of ten animals were collected for micronuclei analysis. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with the substance were in the same range as compared to the negative control groups.

Conclusion

The test article did not induce micronuclei in the bone marrow cells of the mouse, under the test conditions.