Sodium 2,4-diamino-5-(2-hydroxy-5-nitrophenylazo)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 26th of April to the 20th of May, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was 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:

Mordant Brown 033

IUPAC Name:

Mordant Brown 033

Test material form:

other: solid

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.

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 mutageniccompounds.

Metabolic activation:

with and without

Metabolic activation system:

S9

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

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.

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
A Main Assay was performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates wereused at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrientagar plates to establish the number of bacteria in the cultures.
The experiment was 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.

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 Assay.

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:

positive

Remarks:

Dose-related increases in the number of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Moderate to slight toxicity, as indicated by thinning of the background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Dose-related increases in the number of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

Dose-related increases in the number of revertant colonies

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Moderate to slight toxicity, as indicated by thinning of the background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Slight but dose-related increases

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to slight toxicity, as indicated by thinning of the background lawn

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 50.0mg/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. At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. An orange 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.
Dose-related 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. Large and dose-related increases in the number of revertant colonies were observed with TA1537, TA98 and TA100 tester strains, at all concentrations tested up to the most toxic ones, where a slight decrease in the number of revertants was noticed.

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. An orange 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.

Slight toxicity, as indicated by thinning of the background lawn, was observed with TA1535, TA1537 and TA100 tester strains, at the two or three highest dose levels, both in the absence and presence of S9 metabolism.

Large and dose-related increases in the number of revertant colonies were noticed with TA1537, TA98 and TA100, in the absence and presence of S9metabolic activation. Slight but dose-related increases were also observed with TA1535, only in the presence of S9
metabolism.
These increases were greater than twice the concurrent negative control values and fell outside the distribution of the historical control data.
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: No precipitation of the test item was observed

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.

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.

Observation

Toxicity test

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.

At the end of the incubation period, no precipitation of the test item was observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Dose-related toxicity, particularly severe with WP2 uvrA, was observed with all tester strains, in the absence and presence of S9 metabolism. Large increases in revertant numbers were observed with TA1537, TA98 and TA100 tester strains, at all concentrations tested, even at moderate toxic levels, in the absence and presence of S9 metabolic activation.

On the basis of toxicity test results, in Main Assay, using the plate incorporation method, the test item was assayed at specific doses.

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.

Moderate to slight toxicity was observed with TA1535, TA1537 and TA100 tester strains, at the two or three highest concentrations tested, in the absence and presence of S9 metabolism. Dose-related increases in the number of revertant colonies were noticed with TA1537, TA98 and TA100, in the absence and presence of S9 metabolic activation, and with TA1535, only in the presence of S9 metabolism. Since a clear positive response was observed, no further experiment was undertaken.

Results

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