Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 2006 to January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

EC Number:

228-589-1

EC Name:

Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate

Cas Number:

6300-50-1

Molecular formula:

C22H17N5O7S2.2Na

IUPAC Name:

disodium 7-amino-4-hydroxy-3-({4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-2-sulfonate

Test material form:

solid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH (35394 Giessen, Germany)
- Suitability of cells: Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The strain cultures were stored as stock cultures in ampoules with nutrient both + 5% DMSO in liquid nitrogen.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH (35394 Giessen, Germany)
- Suitability of cells: Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in RCC Cytotest Cell Research according to B. Ames and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The strain cultures were stored as stock cultures in ampoules with nutrient both + 5% DMSO in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

No precipitation of the test item occurred up to the highest investigated dose

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility porperties

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µL overlay agar

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for the plate incorporation test.
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity:
In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration

The concentration range included two logarithmic decades. The following concentrations of the active ingredient were tested in experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

DURATION
- Preincubation period: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37ºC for 60 minutes. After pre-incubation 2.0 mL overlay agar (45ºC) was added to each tube. The mixture was poured on selective agar plates.
- After solidification the plates were incubated upside down for at least 48 hours at 37ºC in the dark.

NUMBER OF REPLICATIONS: 3 plates for each strain and dose level

COUNTING
The colonies were counted using the Petri Viewer Mk2 wih the software program Ames Study Manager. Due to intense colour of the test item the revertant colonies were partly counted manually.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not cconsidered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: In experiment strain TA 1535 showed irregular background growth in the control plates, therefore strain TA 1535 had to be repeated with and without metabolic activation

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshits in hte genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella Typhimirium and Eschirichia coli reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella Typhimirium strains TA 1535, TA 1537, TA 98 and TA 100, and Eschirichia colin strain WP2 uvrA.

The assay was performed in five independent experiments. Pre-Experiment/Experiment I, and Experiment II and IIA were performed with and without liver microsomal activation. Pre-Experiment/Experiment I A was performed with metabolic activation, only. Each concentration, including the controls, was tested in triplicate. Since a wrong stock solution was prepared in Experiment II A (results are not reported) this part was repeated (Experiment II B) and was reported as part of experiment II. The test item was tested at the following concentrations of the active ingredient:

Pre-Experiment/EXperiment I and IA: 3; 10; 33; 100; 1000; 2500; and 5000 µg/plate

Experiment II and II B: 10; 33; 100; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxics effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metablilc activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.