Tetrasodium 4-amino-6-[[2,5-dimethoxy-4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-5-hydroxy-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

09 to 19. July 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

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:

Reactive Black 5

IUPAC Name:

Reactive Black 5

Details on test material:

- Name of test material (as cited in study report): Remazol Schwarz B

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: males: 25 to 33 g; females: 22 to 29 g
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: -
- Housing: 5 animals/cage
- Diet: Altromin 1324 rat and mouse diet ad libitum
- Water: tap ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 09 to 19. July 1984

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: freshly 25% suspension in water: 12500 mg TS mixed with water by means of a magnetic stirrer and filled up to 50 mL in a volumetric flask.

Dose level: 2000 and 10000 mg/kg bw
Dose concentration: 250 mg/mL
Dose volume: 8 and 40 mL/kg bw

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

Vehicle control and high dose: 24, 48, and 72 hours
Positive control and low dose: 24 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
250 mg/mL
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000, 10000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

5 per killing time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (Endoxan - batch 0378)

Examinations

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
maximum feasible dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
single administration
sampling 24, 48, and 72 hours after administration (vehicle control and high dose group)

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were stained with May-Grünwald-Giemsa:
- 5 minutes Methanol
- 3 minutes May-Grünwald-solution
- 2 minutes May-Grünwald-solution, diluted with aqua dest. 1 + 1
- 2x rinsed in aqua dest.
- 10 minutes stained with 1 part Giemsa-solution + 6 parts buffer solution pH 7.2 according to Weise
- rinsed in aqua dest.
- air dried
- coating with Entellan


METHOD OF ANALYSIS: (blinded scoring)
1000 polychromatic erythrocytes per animal were scored for incidence of micronuclei. The number of cells with micronuclei was recorded, not the number of individual micronuclei.
1000 mature erythrocytes were scored for incidence of micronuclei as inspection measure
the ratio of polychromatic to normochromatic erythrocytes was determined

Evaluation criteria:

statistically significant difference in number of cells with micronuclei in dose groups compared to respective vehicle control groups

Statistics:

incidence of cells with micronuclei (polychromatic or normochromatic) compared to the vehicle control groups by means of binominal distribution (computer program Diamant) with a level of significance of 95%

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

diarrhea, narrowed lids, squatted posture, discolorations (eyes, extremities, muzzle, tail, ears, skin, urine, feces)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Necropsy findings: dark to light bluish staining of: skin, tongue, peritoneum, fatty tissue, muscles, stomach and intestines, urinary bladder, testes, ovaries, adrenals

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No significant increase in number of cells with micronuclei at 2000 and 10000 mg/kg body weight Remazol Schwarz B. Administration of the TS up to the maximum feasible dose did not result in chromosomal mutations.

Executive summary:

Remazol Schwarz B was administered once orally by gavage as single doses of 0 (vehicle control), 2000 and 10000 mg/kg bodyweight to male and female NMRI mice (5 mice per sex and killing time point). 10000 mg/kg bw had been shown in a preliminary study to be the maximum feasible dose. A positive control group received Endoxan at an oral dose of 50 mg/kg body weight.

Animals were killed 12, 24, or 48 hours after treatment by carbon dioxide asphyxiation. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained. 1000 poly- and 1000 normochromatic erythrocytes were screened for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined.

Under the conditions of the present study, Remazol Schwarz B caused no significant increase in the number of cells with micronuclei in the bone marrow cells of treated animals as compared with the control group. Endoxan however produced a marked increase in number of cells with micronuclei..

The results indicate that, under the conditions of the present study, Remazol Schwarz B is not mutagenic in the in vivo micronucleus test.