Sodium 5-({4-chloro-6-[ethyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-(1-sulfonato-2-naphthyl)diazenyl]naphthalene-2,7-disulfonate

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:

2012-12-12 to 2013-03-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Details on test material:

Name: FAT 40348/F TE
Batch No.: BOP 01-12 (Lot: BS-1109708)
Physical State: solid powder
Colour: red-brown
Density: 1.64 g/cm3 at 20°C
Storage Conditions: at room temperature
pH: 8.5 at 20°C (C° 1 g/L) in water
Molecular weight: 982.96
Melting point: >273°C
Active components: sum of all coloured substance: 82.37%, Main constituents: 46.60%
Date of Analysis: 21 August 2012
Expiry Date: 06 February 2017

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

Test concentrations with justification for top dose:

Pre-experiment for experiment I (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
2.5, 5, 10, 25, 50, 75, 100, 250, 500, 750 µg/mL
Experiment I
without metabolic activation: 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL
and with metabolic activation: 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL
Experiment II
without metabolic activation: 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL
and with metabolic activation: 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: For the pre-experiments for experiment I the test item was dissolved in cell culture medium (MEM + 0% FBS).
For the main experiments and the pre-experiment for experiment II a stock solution of the test item in Aqua ad injectabilia was prepared (tenfold) and processed by sterile filtration. The dilution series was prepared in Aqua ad injectabilia. 10% of the dilution series, and/or Aqua ad injectabilia were added to cell culture medium prior to treatment (resulting in the designated concentrations of the test item).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; 300 µg/mL

Controls 2

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in Aqua ad inj. / medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
Selection time (if incubation with selection agent): about one week

SELECTION 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 evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION 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: ≥ 25 μg/mL; experiment I with S9: ≥ 10 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9:≥ 12 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):
negative

In conclusion, in the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40348/F TE 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 culturedin vitro were exposed to FAT 40348/F TE at concentrations of

- 2.5, 5, 10, 25, 50, 100, 200, 300 and 400 µg/mL (without metabolic activation, Experiment I)

- 0.25, 0.5, 1, 10, 20, 40, 60 and 80 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 750, 1000, 1400, 1800 and 2200 µg/mL (without metabolic activation, Experiment II)

- 0.4, 0.8, 1.5, 3, 6, 12, 25, 50, 75 and 100 µg/mL (with metabolic activation, Experiment II).

FAT 40348/F TE 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 10.9% for 400 µg/mL evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 80 µg/mL with a relative growth of 12.8%. In experiment II without metabolic activation the relative growth was 15.0% for the highest concentration (2200 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 100 µg/mL with a relative growth of 16.7%.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.84 was found at a concentration of 400 µg/mL with a relative growth of 10.9%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.98 was found at a concentration of 20 µg/mL with a relative growth of 29.4%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.00 was found at a concentration of 1000 µg/mL with a relative growth of 35.8%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.78 was found at a concentration of 75 µg/mL with a relative growth of 26.1%.

The positive controlsdidinduce the appropriate response. 

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

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