REAKTIVE GELB F 97494

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 22, 2001 to November 29, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-090-0

EC Name:

-

Cas Number:

477795-15-6

Molecular formula:

C26H24FN8Na3O14S4

IUPAC Name:

Trisodium 2-{2-[1-ethyl-2-hydroxy-4-methyl-6-oxo-5-(sulfonatomethyl)-1,6-dihydropyridin-3-yl]diazen-1-yl}-4-{[4-fluoro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}benzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reaktivgelb F-97494

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

First experiment with 3 h treatment time:

without S9-mix: 500*, 1000, 1500*, 2500, 3000*, 3500* and 5000§ µg/mL
with S9-mix: 500*, 1000, 1500*, 2500, 3000*, 3500* and 5000§ µg/mL

§ = not evaluated because of high toxicity
* = evaluated concentrations

Second experiment with 20 h treatment time:$
without S9-mix: 125, 250, 500, 1000, 1500 and 2000 µg/mL

$=because of mutagenicity in the first experiment analysis of the second experiment was not conducted

Vehicle / solvent:

cell culture medium

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(without metabolic activation)

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION: The cells were treated for 3 h in both the presence and absence of S9-mix. Then, the cells were sampled 20 h after the start of treatment as were the concurrent solvent and positive control cultures. Colcemide was added to each culture 2 h before sampling in order to arrest cell division. Chromosome preparations were made, fixed, stained and examined.

Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCl. Exponentially growing cultures which were more than 50% confluent were trypsinated by an approximately 0.25% (v/v) trypsin solution ready for use. A single cell suspension (culture) was prepared. The trypsin concentration was approxiamtely 0.25% (v/v) in Ca-Mg-free salt solution. Two slides were placed in Quadriperm® dishes which were then seeded with cells to yield 3-4x104 cells/slide. Thus for each dose level and treatment time, duplicate cultures slides were used. The Quadriperm® dishes contained 6 mL MEM with approximately 10% (v/v) FCS. After 48 h, the medium was replaced with one containing approximately 10 % (v/v) FCS and the test substance, or positive control, or solvent and in the presence of metabolic activation additionally 2% (v/v) S9-mix. For the 3 h treatment time, the medium was replaced by normal medium following two rinses. In the second experiment the cells were exposed to the treatment medium without S9-mix for 20 h. 18 h after the start of the treatment, Colcemide was added (approximately 0.05 µg/mL/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared.

Evaluation criteria:

Analysis of metaphases
The slides were coded and 25-100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed and classified. Only metaphases with 22+/-2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap, chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges including intrachanges, chromosome exchanges including intrachanges, dicentrics, pulverization and ring formation. Further the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally the mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is expressed as a percentage. After the metaphases had been evaluated, the code was broken. For each experiment the results from the dose groups were compared with thoseof the control group and the positive control at each sampling time.

Criteria for a valid assay
The assay is considered valid if the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency and positive control induce increases in the mutation frequency which are statistically significant and within the laboratory's normal range
Criteria for clastogenicity
The test substance is classified as clastogenic if it induces a statistically significant increase in the number of phases with aberrations (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test substance is classified as clastogenic if there is a concentration-related increase in the number of phases with aberrations (without gaps). The test substance is classified as nonclastogenic if the tests are negative both with and without metabolic activation.

Statistics:

The Biometry of the results was performed with a one-sided Fisher's exact test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

clastogenicity was observed only at cytotoxic concentration

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3000 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the main experiment cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 21.8% of the solvent control value with and without S9-mix at the 3 h treatment time at the highest concentration tested, 5000 µg/mL. At the 20 h treatment time survival was reduced in a dose-related manner reaching 1.2% of the solvent control value at the highest concentration tested, 2000 µg/mL. After 3 h treatment the mitotic index was reduced (indication of toxicity) significantly after treatment with the highest dose levels with and without S9-mix. After treatment with the test substance there was no relevant increase in the number of polyploidy cells as compared with the solvent controls. The test substance was assessed for its clastogenic potential to induce chromosome aberrations in vitro after short-term treatment (3 h). An enhancement of the aberration rates was observed with and without S9-mix at concentrations of 3000 and 3500 µg/mL inclusive and exclusive gaps. These data were found significantly enhanced in the Fisher's exact-test. This is an indication of heavy chromosomal damage. An enhancement was first observed at a toxic concentration of 3500 µg/mL and therefore an additionally lower toxic concentration of 3000 µg/ml was evaluated. The mutagenic effect was confirmed. Both doses (3000 and 3500 µg/mL) caused a distinct reduction in cell survival and moreover a moderate to distinct decrease in the mitotic index. In summary the test substance induded structural chromosome aberrations at cytotoxic concentrations. On the basis of these findings the evaluation of slides of the longer treatment time (20 h) is not necessary. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Any other information on results incl. tables

SOLUBILITY AND PRELIMINARY TOXICITY TESTING: Test substance was dissolved in cell culture medium. Evaluation of the solubility of that solution in MEM Hank's cell culture medium showed that 5000 µg/mL was the highest practicable concentration and produced no precipitate. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 µg/mL and a range of lower dose levels down to 10 µg/L. These results show that test substance was toxic to the V79 cells in the absence and in the presence of metabolic activation (S9-mix) after 3 and 20 h treatment time. Following treatment for 3 h in the absence and in the presence of S9 metabolic activation, high toxicity was observed at 5000 µg/mL. Survival declined in a dose-related manner reaching 38% of the solvent control value without S9-mix and 35.5% of the solvent control value with S9-mix at the highest dose level, 5000 µg/mL. After 20 h treatment survival was reduced to 64.6% of the solvent control value at a concentration of 1000 µg/mL, while at higher dose levels survival was reduced below the limit of detection. Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test substance solutions did not have a significant effect on these parameters.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was not clastogenic in the absence and presence of metabolic activation at non-cytotoxic concentrations. However, the substance did induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and in the absence of a metabolic activation system.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in V79 cells of the Chinese hamster lung according to OECD Guideline 473, EPA OPPTS 870.5375 and EU Method B.10, in compliance with GLP.

 

The substance was dissolved in cell culture medium and tested at the concentrations ranging from 500-5,000 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 chromosome aberrations, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. The test substance induced a significant and dose-dependent increase in the number of aberrant metaphases and in the number of aberrations inclusive and exclusive gaps with and without metabolic activation at 3 h treatment time at doses of 3,000 and 3,500 µg/mL. However, doses of 3,000 and 3,500 µg/mL caused distinct toxicity in the cell survival test and a moderate to distinct reduction of the mitotic index. Further, the highest concentration of 5,000 µg/mL was not evaluated because of high toxicity.

 

Under the study conditions, the test substance was not clastogenic in the absence and presence of metabolic activation at non-cytotoxic concentrations. However, the substance did induce structural chromosome aberrations in V79 Chinese hamster cells at cytotoxic concentrations in the presence and in the absence of a metabolic activation system.