Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 05 December 2012;
Experiment start date - 12 December 2012;
Experiment completion date - 28 March 2013;
Study completion date - 06 May 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Cas Number:

882878-51-5

Molecular formula:

C39H28CuF2N14Na4016S4

IUPAC Name:

Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: FAT 40825/B TE
Batch No.: BOP 01-12
Physical State: powder
Colour: Black
Storage Conditions: at room temperature
pH: 6 to 8
Density: 1.688 g/cm³ at 19.5 ºC
Purity: 84.8% all coloured organic constituents, main constituent: 63.7%
Date of Analysis: 18 July 2012
Expiry Date: 28 January 2017

Specific details on test material used for the study:

Name: FAT 40825/B TE
Batch No.: BOP 01-12
Physical State: powder
Colour: Black
Storage Conditions: at room temperature
Purity: 84.8% all coloured organic constituents, main constituent: 63.7%
Expiry Date: 28 January 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

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 and 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

Experiment I
without metabolic activation:
10, 25, 50, 100, 1000, 2000, 3500 and 5000 µg/mL
and with metabolic activation:
50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 µg/mL

Experiment II
without metabolic activation:
25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL
and with metabolic activation:
200, 400, 800, 1400, 2000, 2600, 3200, 3800, 4400 and 5000 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: For the pre-experiment the test item was dissolved in cell culture medium (MEM + 0% FBS).

For the main experiments 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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation; 1.0 and 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: at 100 and 1000 μg/mL; experiment I with S9: at 50-250 μg/mL and ≥ 2000 μg/mL; Experiment II without S9: ≥ 500 μg/mL; Experiment II with S9: at 200, 400, 2600 and ≥ 3800 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation: No precipitation of the test item was noted in any of the experiments.

Toxicity:
A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation.

In experiment I without metabolic activation the relative growth was 73.2% for the be highest concentration (5000 µg/mL) evaluated, a slightly reduced growth was observed at concentrations of 100 and 1000 µg/ml, respectively. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 17.9%.

In experiment II without metabolic activation the relative growth was 28.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.3%. Furthermore, in the experiments with metabolic activation toxic effects were observed also in the lowest concentrations (experiment I; between 50 and 250 µg/ml; experiment II: 200 and 400 µg/ml).

Mutagenicity:
In experiment I without metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.
Mutation frequencies with the negative control were found to be 21.34 and 21.00, of the solvent control 23.08 and 13.68 mutants/10E6 cells and in the range of 4.90 to 45.14 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.46 was found at a concentration of 5000 µg/mL with a relative growth of 73.2%.

With metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the
solvent controls.
Mutation frequencies with the negative control were found to be 25.97 and 31.22, of the solvent control 22.13 and 29.55 mutants/10E6 cells and in the range of 14.29 to 54.83 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 3500 µg/mL with a relative growth of 54.6%.
In experiment II without metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies with the negative control were found to be 17.21 and 33.59, of the solvent control 42.28 and 32.74 mutants/10E6 cells and in the range of 27.24 to 61.61 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 2000 µg/mL with a relative growth of 54.2%.
In experiment Il with metabolic activation most mutant values of the negative controls, the solvent controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 10E6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to
the solvent controls.

Mutation frequencies with the negative control were found to be 16.22 and 26.72, of the solvent control 30.57 and 34.82 mutants/10E6 cells and in the range of 4.55 to 57.14 mutants/10E6 cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.75 was found at a concentration of 5000 µg/mL with a relative growth of 34.3%.

DMBA (1.0 and 1.5 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Applicant's summary and conclusion

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item FAT 40825/B 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) conducted according to OECD test guideline 476, V79 cells cultured in vitro were exposed to FAT 40825/B TE at concentrations of

- 10, 25, 50, 100, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment II)

- 200, 400, 800, 1400, 2000, 2600, 3200, 3800, 4400 and 5000 µg/mL (with metabolic activation, Experiment II).

FAT 40825/B 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 73.2% for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 17.9%. In experiment II without metabolic activation the relative growth was 28.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.3%.

 

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.46 was found at a concentration of 5000 µg/mL with a relative growth of 73.2%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 3500 µg/mL with a relative growth of 54.6%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 2000 µg/mL with a relative growth of 54.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.75 was found at a concentration of 5000 µg/mL with a relative growth of 34.3%.

The positive controls did induce 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.