Disodium hydrogen bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(3-)

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:

2012-06-13 to 2012-09-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study performed on the analogue substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)

EC Number:

260-906-9

EC Name:

Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)

Cas Number:

57693-14-8

Molecular formula:

C40H20CrN6O14S2.3Na

Test material form:

other: solid and liquid preparations

Method

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (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- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Sprague Dawley Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

All concentrations used referred to the active components of the test item.Pre-experiment for experiment I without metabolic activation: 3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/mLwith metabolic activation: 1.0, 2.5, 5.0, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/mLExperiment Iwithout metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10 and 20 µg/mLwith metabolic activation: 10, 25, 50, 100, 150, 175, 200, 225, 250, and 275 µg/mLExperiment IIwithout metabolic activation: 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mLwith metabolic activation: 30, 60, 140, 220, 240, 250, 260, 270, 280 and 290 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was dissolved in cell culture medium and diluted prior to treatment

Controls

Controls 1

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activationMigrated to IUCLID6: 300 µg/mL

Controls 2

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activationMigrated to IUCLID6: 1 µg/mL and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in mediumDURATION: 4 h (short-term exposure), 20 h (long-term exposure)Expression time (cells in growth medium): 48-72 hSelection time (if incubation with selection agent): about one weekSELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluatedNUMBER OF CELLS EVALUATED: 400000 cells per flaskDETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.There are several criteria for determining a positive result:-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations; -a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their 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

Remarks:

Experiment I without S9: ≥ 5.0 μg/mL; experiment I with S9: ≥ 225 μg/mL; Experiment II without S9: ≥ 60 μg/mL; Experiment II with S9:≥ 220 μg/mL

Untreated negative controls validity:

valid

Positive controls validity:

valid

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):negativeIn vitro cell gene mutagenicity test was performed on the analogue substance following OECD 476 (HPRT-test). Under the experimental conditions reported, the analogue substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells cultured in vitro were exposed to the analogue substance dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of

- 0.025, 0.05, 0.10, 0.25, 0.50, 1.0, 2.5, 5.0, 10 and 20 µg/mL (without metabolic activation, Experiment I)

- 10, 25, 50, 100, 150, 175, 200, 225, 250 and 275 µg/mL (with metabolic activation, Experiment I)

- 10, 20, 40, 50, 60, 70, 80, 90 and 100 µg/mL (without metabolic activation, Experiment II)

- 30, 60, 140, 220, 240, 250, 260, 270, 280 and 290 µg/mL µg/mL (with metabolic activation, Experiment II).

Analogue substance was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 5.1% for the highest concentration (20 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 275 µg/mL with a relative growth of 19.5%. In experiment II without metabolic activation the relative growth was 20.1% for the highest concentration (100 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 290 µg/mL with a relative growth of 10.1%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 0.80 was found at a concentration of 0.025 µg/mL with a relative growth of 76.4%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.60 was found at a concentration of 250 µg/mL with a relative growth of 31.7%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 100 µg/mL with a relative growth of 20.1%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.98 was found at a concentration of 140 µg/mL with a relative growth of 83.1%.

The positive controlsdidinduce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.