tetrasodium 2-(4-fluoro-6-(methyl-(2-(sulfatoethylsulfonyl)ethyl)amino)-1,3,5-triazin-2-ylamino)-5-hydroxy-6-(4-methyl-2-sulfonatophenylazo)naphthalene-1,7-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 6, 1999 to November 05, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

432-550-6

EC Name:

-

Cas Number:

243858-01-7

Molecular formula:

Hill formula: C25H22FN7Na4O16S5 CAS formula: C25H26FN7O16S5.4Na

IUPAC Name:

tetrasodium 2-({4-fluoro-6-[methyl({2-[2-(sulfonatooxy)ethanesulfonyl]ethyl})amino]-1,3,5-triazin-2-yl}amino)-5-hydroxy-6-[2-(4-methyl-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reaktiv Orange F97-0318

Method

Target gene:

HPRT (i.e., hypoxanthine-guanine phosphoribosyl transferase) locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Source of cells: Cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
-Cell culture medium: MEM (minimal essential medium) with Hankssalts and 25 mM Hepes-buffer
-Experimental conditions in vitro: Approximately 37°C and approximately 4% CO2 in plastic flasks

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

Without S9-mix: 50, 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500* and 3,000*# µg/mL

With S9-mix: 50, 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500* and 3,000*# µg/mL

# = only in the repeat main experiment tested concentration
* = because of high cytotoxicity in the repeat main experiment no mutant selection was performed

Vehicle / solvent:

MEM Hank's cell culture medium

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

Mutagenicity test
Two independent mutation tests were performed. Exponentially growing cultures which were more than 50% confluent were trypsinated by an approximately 0.25% (v/v) trypsin ready for use (mfr. Gibco). A single cell suspension was prepared. Subsequently the cells were replated to determine the mutation frequencyand plating efficiency.

Test procedure
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test substance in the same way as for the preliminary experiment (i.e., described below in any other information on materials and methods incl. tables section). 24 h after seeding of approximately 4,500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (i.e., 5%v/v) medium containing the test substance to which either buffer or S9-mix was added as appropriate. After 4 h the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of approximately 24 h.


The treatment schedule of the mutagenicity test is described below

Day 1: Subculturing of an exponentially growing culture

a) Approximately 4,500 cells in each well of a microtiter plate for determination of the plating efficiency.

b) 6 X 105 - 1 x 106 cells in 175 cm2 flasks with 30 mL medium for the mutagenicity test, one flask per experimental point.

Day 2: Treatment of a) and b) with the test substance in the presence and absence of S9-mix (final protein concentration: approximately 0.3 mg/mL) for 4 h.

Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.

Day 5 or 6: Subculturing of b) in 175 cm2 flasks.

Day 9: Subculturing of b) in five 75 cm2 flasks with culture medium containing 6-thioguanine: Mutant selection (i.e., about 3,00,000 cells/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (i.e., about 400 cells per flask).

Day 16: Fixation and staining of colonies of b) - from subcultures seeded on Day 9.

All incubations were carried out at approximately 37°C and 4% CO2, Staining was performed with approximately 10% (v/v) methylene blue in approximately 0.01% (w/v) KOH solution. Only colonies with more than 50 cells were counted.

Evaluation criteria:

Criteria for a valid assay

The assay is considered valid if the following criteria are met:

- The solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency

- The positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

- The plating efficacy for the solvent control was greater than 50%

Criteria for a positive response

The test substance is classified as mutagenic if:

- It reproducibly induces with one of the test substance concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.

- There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.

- Survival of the responding dose group is at least 30%.

However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results for the test substance is performed off-line with the Mannwhitney- U-Test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 500 µg/mL (with and without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The test substance was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
- No relevant reproducible increases in the mutant colonies or mutant frequency three times over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix.
- The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Any other information on results incl. tables

Mutagenicity

 

Experimental design

-Two independent mutation assays to examine resistance to 6-thioguanine were performed.

 

-In the absence and in the presence of S9 metabolic activation dose levels of 50, 100, 250, 500, 750, 1,000, 1500, 2,000 and 2,500 µg/mL were selected for the main mutation experiment and dose levels of 100, 250, 500, 750, 1,000, 1,500, 2,000, 2,500 and 3,000 µg/mL for the repeat mutation assay. Because of high cytotoxicity with the concentrations of 2,500 and 3,000 µg/mL in the repeat test no mutant selection was performed.

 

-Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test substance solutions did not have any effect on these parameters.

 

Survival after treatment

 

- In the absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 26.2%, respectively 30.7% of the solvent control value in the microtiter plates at the highest dose level tested, 2,500 µg/mL, respectively 3,000 µg/mL.

 

- In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching approximately 49.8%, respectively 46.7% of the solvent control value in the microtiter plates after treatment at the highest dose level, 2,500 µg/mL, respectively 3,000 µg/mL.

Solubility and toxicity

 

-Test substance was dissolved in MEM Hank's cell culture medium. Evaluation of the solubility showed that 5,000 µg/mL was the highest practicable concentration and produced no precipitate.

 

- Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5,000 µg/mL and a range of lower dose levels down to 10 µg/mL.

 

- In the absence and in the presence of S9 metabolic activation survival declined steeply between the three highest concentrations. At a concentration of 1,000 µg/mL survival was reduced to 58.1%,respectively 70.9%of the solvent control value, while at a dose level of 5,000 µg/mL only 3.4%,respectively 2.4%of the solvent control survived.

 

-Based on these results 2,500 µg/mL was selected as the maximum dose level for the main mutation experiment and 3,000 µg/mL for the repeat mutation experiment in both the absence and in the presence of S9-mix. Eight lower concentrations down to 50 µg/mL, respectively 100 µg/mL were also included.

Applicant's summary and conclusion

Conclusions:

The test substance was found to be non-mutagenic in the HPRT assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster lung according to OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 Method, in compliance with GLP.

 

Two independent experiments were conducted both with and without metabolic activation (i.e., S9 mix). The substance was dissolved in cell culture medium and tested at the concentrations ranging from 50-3,000 µg/mL with and without metabolic activation, based on the results of preliminary testing for solubility and toxicity.

 

Positive controls showed a significant increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance did not induce a relevant reproducible increase in the mutant colonies or mutant frequency three times over the range of the solvent control up to the highest investigated dose in two independent experiments, either with or without metabolic activation by S9-mix.

 

Under the study conditions, the test substance was found to be non-mutagenic in the HPRT assay.