Sodium 3-[(1,5-dihydroxy-2-naphthyl)azo]-4-hydroxybenzenesulphonate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: Diamantechtrot BT

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:MEM (minimal essential medium) with Hank's salts and 25 mM Hepes-buffer
- Properly maintained: ye
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix rat

Test concentrations with justification for top dose:

Test groups
a: without metabolic activation: 500, 1000, 2500 and 5000 µg/ml
b: with metabolic activation: 500, 1000, 2500 and 5000 µg/ml

Control groups
negative controls:
a: untreated control
b: cultures treated with the solvent

positive controls:
a: without metabolic activation:EMS (Ethyl methane sulfonate)
b: with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)

Vehicle / solvent:

dissolved in MEM cell culture medium Hank's at appropriate concentrations immediately before use

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

In preliminary toxicity experiments approximately 4500 cells were seeded in each well of a microtiter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. Approx. 24 hours after treatment, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 4500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours 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 approx. 24 hours.

Two independent mutation tests were performed.

Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 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 frequency and plating efficiency (see above).

The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6E5 - E6 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 compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.
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 (about 300 000 cells/flask);
subculturing of b) in two 25 cm2 flasks for plating efficiency (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 approx. 37°C and 4% CO2 , Staining was performed with approx. 10% (v/v) methylene blue in approx. 0.01% (w/v) KOH solution.
Only colonies with more than 50 cells were counted.

Evaluation criteria:

Criteria for a valid assay
- 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
- it reproducibly induces with one of the test compound 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:

MANN-WHITNEY-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:

at 5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Diamantechtrot BT was dissolved in MEM Hank's cell culture medium.
Evaluation of the solubility in MEM cell culture medium showed that 5000 µg/ml was the highest practicable concentration. Microscopic visible precipitation of the test substance was observed at concentrations of 2500 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 10 µg/ml.
These results show that Diamantechtrot BT was not toxic to the V79 cells in the absence of metabolic activation (S9-mix).
In the presence of S9 metabolic activation the results showed only a slight reduction of the extinction at the highest concentration of 5000 µg/ml, where survival was reduced to 74.7 % of the solvent control value.
Based on these results 5000 µg/ml was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Three duplicate lower concentrations down to 500 µg/ml were also included.

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

In the absence of S9 metabolic activation in the first main mutation experiment a dose-related decrease in survival was observed reaching 65.5 % of the solvent control value in the microtiter plates at the highest dose level tested, 5000 µg/ml. This effect was not reproduced in the repeat mutation experiment.
In the presence of S9 metabolic activation survival decreased in a dose-related manner reaching approximately 73 % of the solvent control value in the microtiter plates after treatment at the highest dose level, 5000 µg/ml, in the first main mutation experiment.
In the second mutation experiment survival was slightly decreased reaching 89.5 % of the solvent control value in the microtiter plates at the highest dose level tested, 5000 µg/ml

No significant increases in the mutant colonies or mutant frequency over the range of the solvent control was found of the concentrations used, either with or without metabolic activation by S9-mix in the first main experiment.
Only in the second independent experiment a statistical significant enhancement of the mutation rate over the range of the solvent controls was found without metabolic activation with the concentration of 2500 µg/ml of the test compound. This effect was not reproducible, not dose-dependent and not three fold higher than the solvent control values and therefore considered to be without biological relevance.
The sensitivity of the test system and efficacy of the S9-mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

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

Diamantechtrot BT does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.
Diamantechtrot BT (Batch No. HAS 136401) is therefore considered to be nonmutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Diamantechtrot BT (Batch No. HAS 136401) to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

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

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

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

* = highest concentration tolerated by this test system (OECD - Guideline)

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with metabolic activation.

Precipitation of the test compound was observed only microscopically at the concentration of 2500 µg/ml and above.

Up to the highest investigated dose no relevant and reproducible increase in mutant colony numbers was obtained in two independent experiments. Only in the second independent experiment a statistical significant enhancement of the mutation rate over the range of the solvent controls was found without metabolic activation with the concentration of 2500 µg/ml of the test compound. This effect was not reproducible, not dose-dependent and not three fold higher than the solvent control values and therefore considered to be without biological relevance.

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

In conclusion, Diamantechtrot BT does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

Diamantechtrot BT (Batch No. HAS 136401) is therefore considered to be nonmutagenic in this HPRT assay.