Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: read across from supporting substance

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

421-880-6

EC Name:

-

Molecular formula:

C34H25N11Na2O11S3

IUPAC Name:

disodium 4-amino-6-[[4-(N-(4-((E)-(2,4-diaminophenyl)diazenyl)phenyl)sulfamoyl)phenyl)diazenyl)-5-hydroxy-3-((E)-(4-nitrophenyl)diazenyl)naphthalene-2,7-disulfonate

Method

Target gene:

L5178Y (TK+/-) mouse lymphoma cell line

Metabolic activation:

with and without

Metabolic activation system:

s9-mix

Test concentrations with justification for top dose:

maximum concentration of 625μg/ml

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: N-nitrosodimethylamine

Details on test system and experimental conditions:

Preparation of Test Sample and Positive Controls: All stocks were prepared by diluting the chemical in DMS0 to produce concentrations of 100 times that of the required final concentrations.
Cell Maintenance: A bank of L5178Y (-3.7.2c [TK+/-]) cells (ex Dr D Clive, Burroughs Wellcome, USA) was stored in a liquid nitrogen freezer. The cell stocks have been shown to be free of mycoplasma by enzyme linked immunosorbent assay (ELISA).The cultures were kept at 37°C under an atmosphere of 5% C02 in air, either in a gassing incubator (Forma Scientific) or in a hot room in roller bottles rotated on a roller apparatus (Bellco Glass).

Rationale for test conditions:

A dose-ranging study was performed to determine the concentrations of substance to be used in the main mutation assays. Subsequently, two series of exponentially growing suspension cultures of L5178Y cells were treated in duplicate with the solvent control, positive controls or a range of concentrations of substance S124668 far 4 hours in the presence and absence of S9-mix.
Substance was tested both in the presence and absence of S9-mix up to a maximum concentration of 625μg/ml. After removal of the treatment medium, the cells were counted and a sample diluted to determine the survival immediately after treatment. The remaining cells were then cultured to allow any induced mutants to be detected. During this expression time the growth rate was monitored and, where appropriate, the cells subcultured daily. At the end of the 48 hour expression time, samples were grown in both selective medium (supplemented with trifluorothymidine (TFT)) and non-selective medium, and the results
obtained used to determine the mutant frequency per viable cell. The effect of substance S124668 on the pH and osmolality of the treatment
medium was investigateci as changes in pH and increases in osmolality have been reported to result in increases in mutant frequency (Scott et al, 1991).

Evaluation criteria:

Test data from individual experiments were considered valid if:
(a) the spontaneous mutant frequencies in the presence and absence of S9-mix fell within an acceptable range
(b) the positive controls induced unequivocal positive responses.

A positive response in a valici individual experiment is recorded when statistically significant, dose relateci increases in mutant frequency are
observed across a range of toxicity. Such increases in mutant frequency are not considered indicative of a positive response if the increases occur only at concentrations eliciting excessive toxicity. These increases must also be associateci with increases in the numbers of mutants aver those observed with the concurrent solvent control.
A negative result in a valici experiment is obtained when there is no significant dose relateci increase in mutant frequency compared to the solvent control treated cultures.
A positive response in an individual experiment must be reproducible far the test sample to be considered positive (ie mutagenic) in this assay

Statistics:

A statistical analysis is applied at the discretion of the Study Director in consultation with the Study Statistician. The data were examined by the Study Statistician who, in consultation with the Study Director, considered that a statistical analysis was not necessary

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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

Additional information on results:

In a preliminary dose ranging experiment the maximum concentration of substance in the test medium considered appropriate for testing was determined as 625μg/ml with and without S9-mix as this concentration was the minimum precipitating concentration. No adverse effects on the pH or osmolality of the treatment medium were seen at concentrations in excess of the maximum concentration used in this study.
Treatment with substance produced no significant increases in mutant frequency either in the presence or absence of S9-mix in either experiment.
The positive controls (EMS and NDMA) induced substantial increases in mutant frequency in all mutation experiments, demonstrating the activity of the S9-mix and that the assay was performing satisfactorily in being capable of detecting known mutagens.

Remarks on result:

other: non mutagenic

Applicant's summary and conclusion

Conclusions:

Under the conditions of this assay, substance is non-mutagenic to L5178Y TK+/- cells as determined by selection in trifluorothymidine in both the presence and absence of an auxiliary metabolic activation system (S9-mix), when tested to a concentration (625μg/ml) limited by the solubility of the test sample in the treatment medium

Executive summary:

To assess the mutagenic potential of substance to mammalian cells, L5178Y TK+/- mouse lymphoma cells were treated in vitro with various concentrations of test sample, both in the presence and absence of a rat liver derived auxiliary metabolic activation system (S9-mix). Mutant frequencies were assessed by cell growth in the presence of trifluorothymidine after a 48 hour expression time.

Substance was tested both in the presence and absence of S9-mix in two independent experiments. Both experiments gave negative results in the presence and absence of S9-mix when tested to a maximum of 625μg/ml which

was the minimum precipitating concentration.

It is therefore concluded that, under the conditions of this assay, substance is non-mutagenic to L5178Y TK+/- cells in either the presence or absence of S9-mix when tested to a concentration (625μg/ml) limited by the solubility of the test sample in the treatment medium.