REAKTIV-ORANGE DYPR 1410

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 1998 to 17 March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (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:

432-080-1

EC Name:

-

Molecular formula:

Hill formula: C18H15N3Na2O9S3 CAS formula: C18H17N3Na2O9S3.2Na

IUPAC Name:

Disodium 4-amino-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

microgranulate or well dedusted powder

Details on test material:

Name: Reaktiv-Orange DYPR 1410

Specific details on test material used for the study:

Solubility: dissolved in cell culture medium
Stability in the solvent: not necessary because the test substance was dissolved directly into the test system (MEM)
Concentration of stock solution: 5 mg/ml

Method

Target gene:

Chromosome aberration.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CCX, in 175 cm2 plastic flasks. About 5 x 10s to 1 x 10s cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neornycinsulfate. The cells were subcultured twice a week.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate fraction (S9-mix)

Test concentrations with justification for top dose:

First experiment with 3 h treatment time:
without S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml
with S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml

Second experiment with 20 h treatment time:
without S9-mix: 25#, 50, 100, 250 and 375* μg/ml

* not evaluated because of high toxicity
# not used because higher concentrations were evaluated

Vehicle / solvent:

Dissolved in cell culture medium at appropriate concentrations immediately before use.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Solvent controls: cultures treated with the solvent
Positive controls: a: without metabolic activation: EMS (ethyl methane sulfonate)
b: with metabolic activation: CPA (cyclophosphamide) = Endoxan®
Formulation of test compound: dissolved in cell culture medium at appropriate concentrations immediately before use
Formulation of reference compounds: EMS dissolved in cell culture medium on the day of treatment
final concentration; 1.5 mg/ml (3 h treatment) final concentration: 0.4 mg/ml (20 h treatment)
CPA dissolved in cell culture medium on the day of treatment, final concentration in cell culture medium: 3.0 μg/ml
Source of cells: cell bank of "Genetic Toxicology", HMR Germany, ProTox
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Experimental conditions In vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals using cold sterile solutions at approx. 0 to 4 °C and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix The concentrations of the different cofactors of the S9-mix were:

8 mM MgCI2
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CCX, in 175 cm2 plastic flasks. About 5 x 10s to 1 x 10s cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the
cytogenetic assay. Cell cultures were subjected to the same treatment conditions as in the main experiment. Cytotoxic effects were determined by photometric measurement of V79 cell cultures grown in microwell plates and stained with crystal violet. The relative cell density in the microwell plates was nearly the same as in the Quadriperm® dishes.

The test included the following treatments:

Solvent control : the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 ug/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection

The concentrations for the mutagenicity assay were based on the results of the toxicity experiment.
For non-toxic, freely soluble test compounds, the top dose is 10 mM or 5000 ug/ml according to International testing guidelines,
For relatively insoluble test compounds, that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium after the end of the treatment period. In the case of toxic effects, the highest dose level should reduce the survival rate to approximately 20 - 50 % and/or the mitotic index to approximately 50 % compared with the corresponding solvent control.
For toxic compounds additional concentrations may be included in the treatment series. According to the criteria described above, three adequately spaced dose levels extending over at least one decadic logarithm were evaluated. For each experimental point two cultures were used for each concentration.

Mutagenicity test

Two independent experiments were conducted. The first experiment with 3 hours treatment time of the test substance was performed in the presence and the absence of S9-mix. Cultured celis were seeded onto slides (duplicate culture) then treated for either 3 hours (with and without S9-mix in the first experiment) or for 20 hours (without S9-mix in the second experiment). Celcemide was then added to arrest cell division and the chromosomes were stained and examined. In both assays, cells were sampled 20 hours after the start of treatment. For both assays, at least three dose levels were evaluated for chromosome aberrations. Where negative or equivocal results were obtained, cells were treated and also examined 20 hours after the start of treatment.

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 HCI. Any effects on the osmolality during the study were described in the study report.

Two-day old, exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension (culture) was prepared. The trypsin concentration was approx. 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 2-3 x 10-3 cells/slide. Thus for each dose level and treatment time, duplicate cultures were used. The Quadriperm® dishes contained 5 ml MEM with approx. 10 % (v/v) FCS.

After 48 h, the medium was replaced with one containing approx. 10 % (v/v) FCS and the test compound, or positive control, or solvent and in the presence of metabolic activation additional 2 % ( v/v) S9-mix.
For the 3 hour treatment time, the medium was replaced by normal medium following two rinses. In the repeat experiment the cells were exposed to the treatment medium without S9-mix for 20h.

18 h after the start of the treatment, colcemide was added (approx. 0.05 ug/ml/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared as follows:
The cultures were made hypotonic by adding about 4 ml of approx. 0.075 M potassium chloride solution at around 37 °C, The cells were then incubated for 20 minutes at approx. 37 °C. The next step was the addition of 1.5 ml fixative.

Then the liquid was replaced by 5 ml fixative (methanol: glacial acetic acid, 3 :1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and airdried for 24 hours. The chromosomes were stained as follows:
- staining for 10 minutes in approx. 2 % (w/v) orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan® or Corbit®

Duplicate cultures were prepared from each experimental group,

For both treatment times the solvent and the positive controls were prepared 20 h after treatment in the same way.

Evaluation of data - 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. The chromosomal aberrations were classified as shown in chapter 9.1. 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 inirachangesj dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.

After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.

Evaluation criteria:

Criteria for a valid assay
The assay was considered valid if the following criteria are met:
The solvent control data were 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

Criteria for a positive response
The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test compound is classified as mutagenic if there Is a concentration-related increase in the aberration rale (without gaps).
The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation

Statistics:

The Biometry of the results was performed with a one-sided Fisher - Exact test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and preliminary toxicity testing
Reaktiv-Orange DYPR 1410 was dissolved in cell culture medium. Evaluation of the solubility of that solution in cell culture medium showed that 5000 μg/ml was the highest practicable concentration and produced no macroscopic precipitate. Microscopic visible precipitation was observed at the 3h treatment time from a dose level of 2000 μg/ml up to 5000 μg/ml and at the fixation interval of 20h at a concentration of 1000 μg/ml and above.
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 100 μg/ml.
Following treatment for 3 hours in the absence of S9 metabolic activation, several toxicity was observed at 2000 μg/ml and above. Survival declined in a dose-related manner reaching 51.9 % of the solvent control value at the highest dose level, 5000 μg/ml. After 20 hours treatment survival was reduced to 6.8 % of the solvent control value at a concentration of 500 μp/ml and 12.9 % of the solvent control value at a concentration of 5000 μg/ml.
In the presence of S9 metabolic activation mild toxicity was observed at 2000 μg/ml and above. At a concentration of 5000 μg/ml survival was reduced to 69.4 % of the solvent control value.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Mutagenicity test
In the main experiments cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 40.1 % of the solvent control value without S9-mix at the 3 hours treatment time and 53.9 % in the presence of S9-mix at the highest Concentration tested. 5000 μg/ml. At the 20 hours treatment time survival was reduced in a dose-related manner reaching 17.9 % of the solvent control value without metabolic activation at the concentration of 375 μg/ml.
In the main and in the repeat experiments the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels.
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
The test compound Reaktiv-Orange DYPR 1410 was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments.
There was an enhancement of the aberration rates at a non-toxic concentration of 2500 μg/ml at the 3 h treatment time with and without S9-mix. Also at the sampling time of 3 h with and without S9-mix at the toxic concentration of 5000 μg/ml the aberration rates were enhanced. These data were found significantly enhanced in the Fisher's exact-test. Because of the lower dose range at the 20 h treatment time no enhancement of the aberration rates was found. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Remarks on result:

not determinable because of methodological limitations

Any other information on results incl. tables

In the chromosomal aberration test strong positive effects were obtained with and without S-9 mix at doses which induced precipitations. However, it is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system -with a very limited amount of phase 2 enzymes- caused by the Michael Addition at the vinyl sulphone structures. However, it was shown in many (>100) studies that this effect is not noted in in-vivo studies (as is the case with the substance here). One explanation is that the cause for this is that in vivo, the relation of the Michael Addition and the GSH is much in favour for the repair system. As such, these effects are not noted. In addition, the chromosome aberration test was only positive at the 3 h at doses and precipitates were seen. It is well-known that these precipitates can cause a false positive reaction. Therefore, the chromosome aberration text in vitro cannot be assessed as unequivocally positive.

Applicant's summary and conclusion

Conclusions:

Reaktiv-Orange DYPR 1410 was apparently cytogenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described in this report.
It is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system, with a very limited amount of phase 2 enzymes, caused by the Michael Addition at the vinyl sulphone structures. However, it was shown in many (>100) studies that this effect is not noted in in-vivo studies (as is the case with the substance here). One explanation is that the cause for this is that in vivo, the relation of the Michael Addition and the GSH is much in favour for the repair system. As such, these effects are not noted.

Executive summary:

The study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 473 "Genetic Toxicology: In VitroMammalian Chromosome Aberration Test". U.S. EPA: OPPTS 870.5375 Health Effects Test Guidelines In Vitro Mammalian Chromosome Aberration Test and EEC Directive 92f69, L3S3 A, Annex B. 10., p. 148 -150. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

In this study the potential of Reaktiv-Orange DYPR 1410 to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lungin vitro.For each experiment duplicate cultures were used for each concentration,

 

The test compound was dissolved in cell culture medium and tested at the following concentrations:

 

First experiment with 3 h treatment time:

without S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml

with S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml

 

Second experiment with 20 h treatment time:

 

without S9-mix: 25#, 50, 100, 250 and 375* μg/ml

 

*          not evaluated because of high toxicity

#         not used because higher concentrations were evaluated

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced a distinct lowering of the mitotic index. Higher concentrations were not applied because of the 5000 μg/ml limitation (OECD guideline).

At the 3h sampling time the concentrations of 2500 and 5000 µg/ml with and without metabolic activation the test compound induced a significant increase in the number of chromosome aberrations.

 

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, Reaktiv-Orange DYPR 1410 appears to induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

In the chromosomal aberration test strong positive effects were obtained with and without S-9 mix at doses which induced precipitations. However, it is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system -with a very limited amount of phase 2 enzymes- caused by the Michael Addition at the vinyl sulphone structures. However, it was shown in many (>100) studies that this effect is not noted in in-vivo studies (as is the case with the substance here). One explanation is that the cause for this is that in vivo, the relation of the Michael Addition and the GSH is much in favour for the repair system. As such, these effects are not noted. In addition, the chromosome aberration test was only positive at the 3 h doses and precipitates were seen. It is well-known that these precipitates can cause a false positive reaction. Therefore, the chromosome aberration text in vitro cannot be assessed as unequivocally positive.