Disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From March to April, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP. Nevertheless the test is performed on analogue substance. Justification for Read Across is detailed in the endpoint summary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 4, 1984

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 03

IUPAC Name:

Similar Substance 03

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of testing laboratory allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium.
Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 1.0; 3.0; 10.0; 15.0; 25.0 and 50.0 µg/ml
with S9 mix: 3.0, 30.0, 100.0; 150.0; 200.0 and 250.0 µg/ml
Experiment II:
without S9 mix: 1.0; 20.0; 30.0; 40.0; 45.0 and 50.0 µg/ml
with S9 mix: 3.0; 100.0; 200.0; 230.0; 240.0 and 250.0 µg/ml
Experiment III:
without S9 mix: 1.0; 5.0; 10.0; 15.0; 20.0 and 25.0 µg/ml
with S9 mix: 3.0; 50.0; 100.0; 150.0; 180.0 and 200.0 µg/ml

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the solvent was chosen according to its solubility properties and its non-toxicity for the cells. The final concentration of DMSO in the culture medium did not exceed 1 % v/v.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

Without metabolic activation: EMS; with metabolic activation: DMBA.

Details on test system and experimental conditions:

PRE-TEST on TOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment. The following method was used in the pre-test:
XTT-Assay. The XTT-assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye by hydrogenase activity in active mitochondria. 18 - 20 h after treatment with the test article the XTT-assay was initiated by adding a mixture of XTT-labelling reagent with an electron coupling reagent (PMS). After 4 h of incubation the absorption was determined at 450 nm (690 nm reference) using an ELISA reader. The viabilities of the cells are calculated as percentages of the solvent controls and reported as tables.

DOSE SELECTION
In the pre-test of toxicity (XTT-assay) the extinction (measured at 450/690 nm) was reduced after treatment with concentrations higher than 30.0 µg/ml (without S9 mix) and slightly reduced at 100 µg/ml in DMSO (with S9 mix).
Experiment I was performed with six concentrations ranging from 1.0 to 50.0 µg/ml without and from 3.0 to 250.0 µg/ml with metabolic activation. The highest concentration (50 µg/ml) without S9 could not be assessed in this experiment due to strong toxic effects.
Strong toxic effects occurred in the second experiment already at 40 µg/ml and above without metabolic activation and at 200 µg/ml or higher with metabolic activation. In experiment III most evaluated concentrations were close to the limit of toxicity to detect possible mutagenic effects in the toxic range.

EXPERIMENTAL PERFORMANCE
Seeding
Threedays old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution.
The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHC03 350 mg
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid).
The cell suspension was seeded into plastic culture flasks. Approximately 1.5x10^6 (single culture) and 5x10^2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.

Treatment
After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 µl/ml S9 mix. After 4 h this medium was replaced with complete medium after two washing steps with "saline G".
The "saline G" solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HP04x7H20 290 mg, KH2P04 150 mg,.
pH is adjusted to 7.2.

Experimental scheme
Day 1: Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 3.2 (experiment I),. 11.1 (experiment II) and 4.8 (experiment 3) mutants per 10^6 cells.
a) About 500 cells in 5 ml medium/25 cm2-plastic-flask for cloning efficiency; in duplicate per experimental point
b) 1x10^6 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)
experiment I
Day 5: Sub-culturing of b) in 175 cm2-plastic-flasks 1.5x10^6 cells in 30 ml medium/175 cm2-plastic-flasks
experiment II
Day 6: see day 5
Day 8: Fixation and staining of colonies in a)-flasks determination of concentration-related cloning efficiency
Day 9: Sub-culturing of b) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 3-5x10^5 cells/flask);
sub-culturing of b) in two 25 cm2-flasks for cloning efficiency (about 500 cells/flask)
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (cloning efficiency).
Day 18: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection).
The third experiment was performed according to the schedule of experiment I.
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.

Evaluation criteria:

A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points.
A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations.
The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10^ cells) a concentration-related increase of the mutations within this range has to be discussed.

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

Additional information on results:

Without metabolic activation strong toxic effects occurred already at concentrations as low as 25.00 µg/ml. With metabolic activation, the concentration of the test article could be increased up to 250 µg/ml in experiment I and 200 µg/ml in experiment II. Precipitation of the test article occurred at concentrations of 100 µg/ml and above.
The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested.
The number of mutant colonies was not increased compared to the frequency of spontaneous mutations at any concentration of the test article. There was also no indication of a concentration depend increase of mutant colonies.
In all experiments of this study (with and without S9 mix) the range of the negative controls was from 0.6 up to 18.7 mutants per 10^6 cells; the range of the groups treated with the test article was from 4.1 up to 18.9 mutants per 10^6 cells.

EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

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

Test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

Method

The study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in three independent experiments, using identical procedures, both with and without liver microsomal activation. Due to unexpectedly strong toxic effects an additional experiment was required to complete the data of experiment II. In the absence of metabolic activation the concentration range was limited by toxicity, in the presence of metabolic activation by solubility of the test article.

The test article was tested with the following concentrations:

Experiment I:

without S9 mix: 1.0; 3.0; 10.0; 15.0; 25.0 and 50.0 µg/ml

with S9 mix: 3.0, 30.0, 100.0; 150.0; 200.0 and 250.0 µg/ml

Experiment II:

without S9 mix: 1.0; 20.0; 30.0; 40.0; 45.0 and 50.0 µg/ml

with S9 mix: 3.0; 100.0; 200.0; 230.0; 240.0 and 250.0 µg/ml

Experiment III:

without S9 mix: 1.0; 5.0; 10.0; 15.0; 20.0 and 25.0 µg/ml

with S9 mix: 3.0; 50.0; 100.0; 150.0; 180.0 and 200.0 µg/ml

Precipitation of the test article occurred at concentrations of 100 µg/ml and above in all three experiments.

Results

According to the pre-test on toxicity the concentration ranges were selected to yield concentration-related toxic effects. The highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.

Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in all independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusion

In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, test substance is considered to be non-mutagenic in this HPRT assay.