Reaction mass of (disodium 4-[[4-(acetylmethylamino)-2-sulphonatophenyl]amino]1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium sulphate and sodium chloride)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Full report according to Guideline and GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sandolan Blue E-HRL

IUPAC Name:

Sandolan Blue E-HRL

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/ml
with S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/m
Experiment II:
without S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/m
with S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/ml
* not evaluated, culture not continued;

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none /medium

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding
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 (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400mg
Glucose 1000 mg
NaHC03 350mg

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/1 EDTA (ethylenediamine tetraacetic acid).
The cell suspension was seeded into plastic culture flasks (Greiner, D-72632 Frickenhausen).
Approximately 1. 5 x 1E06 (single culture) and 5 x 1E02 cells (in duplicate) were seeded in MEM with 10% FCS (complete medium) for the determination of mutation rate and toxicity, respectively (see experimental scheme).

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 following two washing steps with "saline G" .
The Saline G solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400mg
Glucose 1100 mg
Na2HP04x1H20 290mg
KH2P04 150mg

the pH was adjusted to 7.2.

Experimental Scheme:
Segment a): Procedure for determination of toxicity
Segment b): Procedure for determination ofmutation rates
Day 1: Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 1.7 (experiment I) and 6.6 (experiment II) mutants per 10E6 cells.
a) About 500 cells in 5 ml medium/25 cm2 -plastic-flask for cloning efficiency; in duplicate per experimental point
b) About 1.5xl06 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: Subculturing ofb) in 175 cm2-plastic-flasks 1.5x106 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: Subculturing ofb) in five 80 cm2-plastic-flasks containing selective medium:
mutant selection (about 3-5x105 cells/flask);
subculturing ofb) 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 1 7: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection).

The cultures were incubated at 37° C in a humidified atmosphere with 4.5 % C02. The colonies were stained with 10% methylene blue in 0.01 % KOH solution (E. MERCK, D-64293 Darmstadt, F.R.G.).
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope (Nikon, D-40407 Dusseldorf, F.R.G.).

Evaluation criteria:

Acceptability of the Assay

The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 106 cells found in the negative and/or solvent controls fall within the laboratory historical control data range: 1 - 32 mutants/10E6 cells.
b) the positive control substances must produce a significant increase in mutant colony frequencies.
c) the cloning efficiency (absolute value) of the negative and/or solvent controls must exceed s0 %.

The data of this study comply with the above mentioned criteria


Evaluation of Results

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 at 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:
A test article is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
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 obsenred.
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 (1 - 32 mutants per 10E6 cells) a concentration-related increase of the mutations within this range has to be discussed.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce gene mutations in the HPRT in V79 cells in vitro with and without metabolic activation.

Executive summary:

The test article was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments using identical procedures, both with and without liver microsomal activation.

The test article was tested with the following concentrations:

Experiment I:

without S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/mL

with S9 mix: 30.0; 100.0*; 300.0, 1000.0, 3000.0 and 5000.0 µg/mL

Experiment II:

without S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/mL

with S9 mix: 30.0*; 300.0; 1000.0, 3000.0 and 5000.0 µg/mL

* not evaluated, culture not continued;

Up to the highest test concentration no visible precipitation occurred.

Strong toxic effects, evident as a reduction in the cloning efficiency of the cells occurred in both experiments at 5000 µg/mLw

ithout metabolic activation. In the presence of metabolic a

ctivation only slight toxic effects were observed at the maximal concentration. Up to the highest investigated concentration no relevant and reproducible increase in mutant colony numbers was observed, neither in the presence nor in the absence of metabolic activation.

In the first experiment without metabolic activation unusually low numbers of spontaneous mutant colonies occurred in the solvent control due to statistical fluctuations at such small numbers. This effects results in a colony count above 3 times the number of mutant colonies of the corresponding solvent control at almost all concentrations throughout the experiment. This increase was judged to be biologically irelevant since it is based upon the unusually low colony count of the solvent control. Furthermore, the absolute numbers of colonies are low at all concentrations of the test article and remain well within the range of the solvent controls of the

experiments.

In this study in both experiments (with and without S9 mix) the range of the solvent controls was from 1.3 up to 11.8 mutants per 10E6 cells; the range of the groups treated with the test article was from 2.1up to 17.5 mutants per l0E6 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.

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