Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From the 12th of May to the 13th of June, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test conducted according to an internationally accepted test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

EC Number:

274-403-7

EC Name:

Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Cas Number:

70210-06-9

Molecular formula:

C31H25N5O6S2.2Na

IUPAC Name:

disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate

Test material form:

solid: particulate/powder

Method

Target gene:

Reverse mutation assays employ bacterial strains which are already mutant at a locus whose phenotypic effects are easily detected.
The Salmonella tester strains have mutations causing dependence on a particular amino acid (histidine) for growth.
The ability of test items to cause reverse mutations (reversions) to histidine-independence can easily be measured.
The E. coli tester strains of the WP2 series are similarly mutant at the tryptophan locus.

Since for azo-dyes and diazocompounds, a reductive metabolic activation system could be more appropriate than the standard liver metabolizing system, in Main Assay II, the pre-incubation method was used and the metabolising system was prepared with S9 liver fraction from uninduced hamsters, supplemented with flavin mononucleotide cofactor (Prival et al. 1984).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens.
In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds.
The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface
of the bacterial cell wall and increases permeability to certain classes of chemicals.
All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens.
TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens.
In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

S9 and Privall

Test concentrations with justification for top dose:

5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.

Vehicle / solvent:

Sterile water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and water

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Absence S9

Controls 2

Untreated negative controls:

yes

Remarks:

sterile water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Presence of S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanhtracene, Trypan Blue

Remarks:

Presenceof S9, Reductive metabolic system

Details on test system and experimental conditions:

Permanent stocks of these strains are kept at -80°C in the laboratory.
Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Histidine requirement
No Growth onMinimal plates+Biotin. Growth on Minimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates. Growth onMinimal plates+Tryptophan.
uvrA, uvrB
Sensitivity to UV irradiation.
rfa
Sensitivity to Crystal Violet.
pKM101
Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

MEDIA
Nutrient Broth
Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar
Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar
Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-BonnerMedium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar
"Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).

S9 tissue homogenate
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics: Rat, Strain Sprague Dawley, Liver, Inducing Agents Phenobarbital – 5,6-Benzoflavone, Producer MOLTOX,Molecular Toxicology, Inc., Batch Number 3488
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared to 10.0 mL.

S9 mix Privall modification (Main Assay II) Species Syrian hamster, Strain GSH, Tissue Liver, Inducing Agents None, Producer MOLTOX,Molecular Toxicology, Inc., Batch Number 3342. The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared to 10.0 mL.

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assay. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included.
Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
MAIN ASSAY
Three Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and diluti ons of the bacterial cultures were plated on nutrientagar plates to establish the number of bacteria in the cultures

Main Assay I and III were performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.
Main Assay II was performed using a pre-incubation method. The components were added in turn to an empty test-tube.
The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

Incubation and scoring
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours, in the preliminary toxicity test, and immediately scored by counting the number of revertant colonies on each plate, in the Main Assays.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.
The test item induced two-fold and more increases in the number of revertant colonies, both in the absence and presence of S9 metabolism. A statistically significant dose-related relationship was indicated. Since a clear positive response was observed, no further experiment was undertaken.

Statistics:

The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a +bx

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Dose related increases in the number of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A reddish dose-related colouring, which did not interfere with scoring neither with the evaluation of the background lawn,was observed. The number of revertant colonies exceeded the upper confidence limit

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Dose related increases in the number of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

A reddish dose-related colouring, which did not interfere with scoring neither with the evaluation of the background lawn,was observed. The number of revertant colonies exceeded the upper confidence limit

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

A moderate thinning of the background lawn but a total lack of revertant numbers

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

A moderate thinning of the background lawn but a total lack of revertant numbers

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight toxic effect, as indicated by thinning of the background lawn, at the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Large and dose related increases in revertant numbers

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Dose related increases in the number of revertant colonies. A reddish dose related colouring, which did not interfere with scoring.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Dose related increases in the number of revertant colonies. A reddish dose related colouring, which did not interfere with scoring.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility
Solubility of the test item was evaluated in a preliminary trial using sterile water for injection.
This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0 mg/mL (expressed in terms of active ingredient). This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Toxicity test
The test item was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. A reddish dose-related colouring, which did not interfere with scoring neither with the evaluation of the background lawn, was observed, at the end of the incubation period, with any tester strain, in the absence and presence of S9 metabolism.
No toxicity, as indicated by thinning or total lack of the background lawn and microcolony formation, was observed with all tester strains, in the absence or presence of S9 metabolism. A slight increase increase in the number of revertant colonies were observed with TA100, in the absence of S9 metabolism, and with WP2 uvrA tester strain, in the presence of S9 metabolic activation, only at the highest dose level.
A more pronounced mutagenic effect was observed with TA100 tester strain in the presence of S9 metabolism, where a dose related increase in revertant colonies was noticed at higher dose levels.

Main assays
Three Main Assays were performed.
On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate.
At the end of the incubation period, no toxicity, as indicated by thinning of the background lawn and/or reduction in revertant numbers, neithe r increases in revertant numbers were observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.
Dose related increases in the number of revertant colonies were noticed with TA1537 and TA100 tester strains, both in the absence and presence of S9 metabolic activation. The number of revertant colonies exceeded the upper confidence limit of the historical control range,at higher dose levels.
As the increases in revertant numbers, observed in Main Assay I, were not clearly positive, a pre-incubation step using a reductive metabolic activation system, was included for all treatments of Main Assay II. The test item was assayed at the following dose levels: 5000, 2500,1250,625 and 313 µg/plate.
A moderate thinning of the background lawn but a total lack of revertant numbers, were observed with TA98 and TA100 tester strains, at all dose levels, both in the absence and presence of S9 metabolism.
A slight toxic effect, as indicated by thinning of the background lawn,was noticed with TA1535,at the highest concentration tested,both in the absence and presence of S9 metabolic activation.
Large and dose related increases in revertant numbers were observed with TA1537 tester strain,both in the absence and presence of S9 metabolic activation.
Due to the high toxicity observed using the Prival modification method, a Main Assay III, using the plate incorporation method using the standard metabolic activation system, was performed. This experiment was performed with TA1537 and TA100 tester strains,in order to confirm the observed increases in revertant colonies.
The following dose levels were selected in order to investigate more closely those doses of the test item most likely to exhibit a mutagenic response: 5000, 3330, 2220, 1480 and 988 µg/plate.
Dose related increases in the number of revertant colonies were noticed with TA1537 and TA100 tester strains,both in the absence and presence of S9 metabolic activation. At higher dose levels, the number of revertant colonies fell out the normal distribution of historical controldata. A reddish dose related colouring, which did not interfere with scoring neither with the evaluation of the background lawn, was observed in all Main Assays, at the end of the incubation period,with all tester strains,in the absence and presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items,indicating that the assay system was functioning correctly.

Remarks on result:

other: Main Assay I

Applicant's summary and conclusion

Conclusions:

The test item induced reverse mutation in Salmonella typhimurium tester strains both in the absence and presence of S9 metabolism, under the
reported experimental conditions.

Executive summary:

Method

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone, in Main Assay I and Main Assay III, and liver S9 fraction from uninduced hamsters(reductive metabolic activation system with Prival modification), in Main Assay II.

The test item was used as a solution in sterile water for injection and, as requested by the Sponsor, concentrations were expressed in terms of active ingredient.

Toxicity test

5000, 1580, 500, 158 and 50.0 µg/plate, standard metabolic activation system.

Main Assay I

5000, 2500, 1250, 625 and 313 µg/plate, reductive metabolic activation system.

Main Assay II

5000, 2500, 1250, 625 and 313 µg/plate, reductive metabolic activation system.

Main Assay III

TA1537-TA100, 5000, 3300, 2220, 1480 and 988 µg/plate, standard metabolic activation system.

Results

Toxicity test

No toxicity was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. A slight increase in the number of revertant colonies was observed with TA100, in the absence of S9 metabolism, and with WP2uvrA tester strain, in the presence of S9 metabolic activation, only at the highest dose level. A more pronounced mutagenic effect was observed with TA100 tester strain in the presence of S9 metabolism, where a dose related increase in revertant colonies was noticed at higher dose levels.

Main Assay I

At the end of the incubation period, no precipitation of the test item neither toxic effects were observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Dose related increases in the number of revertant colonies were noticed with TA1537 and TA100 tester strains, both in the absence and presence of S9 metabolic activation. With TA1537,the revertant numbers were greater than twice the concurrent negative control, at the highest dose level.

Main Assay II

No precipitation of the test item was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Severe toxicity was observed with TA98 and TA100 tester strains, at all dose levels, both in the absence and presence of S9 metabolism,while a slight toxic effect was noticed with TA1535, at the highest concentration tested, both in the absence and presence of S9 metabolic activation. Increases in revertant numbers were observed with TA1537 tester strain, both in the absence and presence of S9 metabolic activation, confirming the positive results obtained in the previous assay.

Main Assay III

No precipitation of the test item was observed, at the end of the incubation period, with any tester strain, at any dose level, in the absence or presence of S9 metabolism. Once again, treatments of TA1537 tester strain, both in the absence and presence of S9 metabolism, produced dose-related increases, which were greater than twice the control value at the highest dose level. Slight, but dose related and reproducible increases observed with TA100 tester strain, were al most certainly a further evidence of this mutagenic effect.

Conclusion

It is concluded that the test item induces reverse mutation in Salmonella typhimurium, both in the absence and presence of S9 metabolism, under the reported experimental conditions.