reaction mass of mono to tetra(lithium and/or sodium)3-amino-10-[4-(4-amino-3-sulfonatoanilino)-6-[methyl-(2-sulfonatoethyl)amino]-1,3,5-triazin-2-ylamino]-6-13-dichlorobenzo[1,2-B:4,5-B']di[1,4]benzoxazine-4,11-disulfonate; mono to tetra(lithium and/or sodium)3-amino-10-[4,6-bis(4-amino-3-sulfonatoanilino)-1,3,5-triazin-2-ylamino]-6-13-dichlorobenzo[1,2-B:4,5-B']di[1,4]benzoxazine-4,11-disulfonate; mono to penta(lithium and/or sodium)10,10´-diamino-6,6',13,13´-tetrachloro-3,3'-[6-[methyl-(2-sulfonatoethyl)amino]-1,3,5-triazin-2,4-diyldiimino]bis[benzo[1,2-B:4,5-B']di[1,4]benzoxazine-4,11-disulfonate; mono to hepta(lithium and/or sodium)10-amino-6,6',13,13'-tetrachloro-10´[4-(4-amino-3-sulfonatoanilino)-[6-methyl-(2-sulfonatoethyl)amino]-1,3,5-triazin-2,4-diimino]bis[benzo[1,2-B:4,5-B']di[1,4]benzoxazine-4,11-disulfonate; mono to hepta(lithium and/or sodium)10,10'-diamino-6,6',3,3'[(2-sulfonato)-1,4-phenylenediiminobis[6-methyl-(2-sulfonatoethyl)amino]-1,3,5-triazin-2,4-diyldiimino]bis[benzo[1,2-B:4,5-B']di[1,4]benzoxazine-4,11-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Registered substance

IUPAC Name:

Registered substance

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background:
yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver, phénobarbital/ß-naphthoflavone.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 15.6 ... 500 µg/ml
Concentration range in the main test (without metabolic activation): 9.4 ... 300 µg/ml
Concentration range in the main test (without metabolic activation): 18.75 ... 600 µg/ml

Vehicle / solvent:

distd. water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Experimental Scheme:
Experiment I and II:
Segment a): Procedure for determination of toxicity
Segment b): Procedure for determination of mutation rates
day 1
Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 1.9 mutant colonies per 10^6 cells
(Experiment I) and 6.5 mutant colonies per 10^6 cells (Experiment II).
a) About 500 cells in 5 ml medium/25 cm2-plastic-flask for cloning efficiency; in duplicate per experimental point
b) About 1 5x10^6 cells in 30 ml medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point
day 2 Treatment of a) and b)
day 5 Subculturing of b) in 175 cm 2-plastic-flasks 1 5x10^6 cells in 30 ml medium/175 cm2-plastic-flasks
day 8/10 Fixation and staining of colonies in the flasks used to determine toxicity (a) determination of concentration-related cloning efficiency
day 9 Subcultivation of (b) in five 80 cm 2-plastic-flasks containing selective medium to select mutant colonies (about 3-5 x10^5 cells/flask) and in two 25 cm 2-
flasks without selective medium to determine the cloning efficiency (about 500 cells/flask)
day 16/17 Fixation and staining of the colonies to determine the cloning efficiency and the number of mutant colonies.
The cultures were incubated at 37° C in a humidified atmosphere with 4.5 % CO2. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
SELECTION AGENT (mutation assays): 6-Thioguanine
STAIN: 10 % methylene blue in 0.01 % KOH solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: >1.5x10^ 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10^6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 1996 - 1997.
b) the positive control substances must produce a significant increase in mutant colony frequencies.
c) the cloning efficiency (absolute value) of the negative and/or solvent controls must exceed 50 % .

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not
available

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

not subject for clasification;