Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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:

Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride

EC Number:

916-466-1

IUPAC Name:

Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride

Details on test material:

Identification : C059 (Basic Blue 99)
Batch number : 74/75
3-[(Bromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (main) : 62.8a/a%
3-[(Dibromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (E) : 11.8a/a% 3-[(Bromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (F) : 8.4a/a%
3-[(Bromo-5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (J) : 4.1a/a% 3-[(8-Amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (A) : 1.7a/a% 3-[(Sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (B) : 1.8a/a%
3-[(Dibromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (G): 2.1a/a%
3-[(Sulpho-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (O) :1.0a/a%
3-[(Bromo-sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (N) : 0.7a/a%
Tribromo-8-amino-5-hydroxy-1,4-naphthoquinone (L) : 2.6a/a%
Chloride ion : 20.2%
Sulphate ion : 0.5%
Acetate ion : 2.0%
Zinc ion : 6.6%
Water : 7.9%

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Deionised water

Details on exposure:

On the day of the experiment, the test item was dissolved in deionised water.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Single treatment

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1.25 mg/kg bw
Basis:
other: administered intraperitoneally, sampling time 24 hours

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2.5 mg/kg bw
Basis:
other: administered intraperitoneally, sampling time 24 hours

Doses / concentrations 3

Remarks:

Doses / Concentrations:
5.0 mg/kg bw
Basis:
other: administered intraperitoneally, sampling time, 24 hours

Doses / concentrations 4

Remarks:

Doses / Concentrations:
5.0 mg/kg bw
Basis:
other: administered intraperitoneally, sampling time 48 hours

No. of animals per sex per dose:

Six/sex/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control : Cyclophosphamide

Examinations

Tissues and cell types examined:

Bone marrow

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group. The positive control showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. The test item is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

The study was performed to investigate the potential of Basic Blue 99 to induce micronuclei in polychromatic erythrocytes in the bone marrow of the mouse. The test item was formulated in deionised water which was also used as the vehicle control. The volume administered intraperitoneally was 10 mL/kg bw, 24 and 48 hours after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes per animal were scored for micronuclei. Test item dose levels of 1.25, 2.5, 5.0 mg/kg bw (24 hours preparation interval) and 5.0 mg/kg bw (48 hours preparation interval) were investigated. As estimated by pre-experiments 5.0 mg/kg bw was the highest applicable dose without significant effects on the survival rates but with clear signs of toxicity. At the next higher dose (7.5 mg/kg bw) one female died. After treatment with the test item the number of polychromatic erythrocytes was not substantially decreased as compared to the mean value of polychromatic erythrocytes of the vehicle control thus indicating that Basic Blue 99 did not exert any cytotoxic effects in the bone marrow. The bedding of the treated animals was stained blue, most probably resulting from the test item content in urine. This indicates the systemic distribution of the test item and thus confirms bioavailability of the test item. In comparison to the corresponding vehicle controls there was no biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Cyclophosphamide (40 mg/kg bw) administered intraperitoneally was used as a positive control which showed a substantial increase of induced micronucleus frequency. Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Basic Blue 99 is considered to be non-mutagenic in this micronucleus assay.