Reaction products of diazotized Dodecyl Aniline coupled with 8-acetylamine-1-hydroxynaphthalen-3,6-disulphonic Acid, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

EC Number:

946-819-5

Molecular formula:

Not applicable for UVCB substance

IUPAC Name:

disodium 3-[(1E)-2-(4-dodecylphenyl)diazen-1-yl]-5-acetamido-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In Main Assay I: 5000, 2500, 1250, 625, 313 and 156 µg/plate.
Toxicity test: 2500, 791, 250, 79.1, 25.0 µg/plate

Vehicle / solvent:

- Vehicle and solvent used:water, DMSO

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 Standard metabolic activation system

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene 1, 2.5 and 30 µg/plate in DMSO; Trypan blue 400 µg/plate

Remarks:

with S9 Reductive metabolic activation system

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
The following growth media were used:
-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-Bonner Medium 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, 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 ml).

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1537, TA 1535, TA98, TA100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY
An opaque formulation with slight precipitation, suitable for treatment but not for serial dilutions, was obtained in water for injection at 25.0 mg/ml, following vortexing and sonication at 37°C for approximately 30 minutes. An opaque formulation without any precipitation was obtained at 12.5 mg/ml and was considered suitable for serial dilutions.

TOXICITY TEST
No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation. Plates treated with the test item presented a dose dependent light red colour of the agar, which did not interfere with the scoring of colonies.
Neither toxicity, nor relevant increases in revertant colonies were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

MAIN ASSAYS
Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. Based on these results, aMain Assay II was performed using the Prival modification method and the same concentration range. Neither toxicity, nor relevant increases in revertant numbers were observed at any dose level, with any tester strain, in the absence or presence of S9 metabolic activation.
In both assays, plates treated with the test item presented a dose dependent light red colour of the agar (red-yellow in the presence of the Prival modified metabolic system), which did not interfere with the scoring of colonies.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.

EVALUATION
Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. 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.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

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 according to the OECD 471 (1997). 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 (standard metabolic activation) in Main Assay I, 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/suspension in sterile water for injection. The test item was assayed in the toxicity test at a maximum concentration of 2500 µg/plate and at four lower concentrations. 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. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in order to evaluate a potential mutagenic effect up to cytotoxic or insoluble concentrations, in the Main Assays an additional dose level of 5000 µg/plate was employed. In Main Assay I, using the plate incorporation method, the test item was assayed at six dose levels from 156 to 5000 µg/plate. As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was carried out. Based The experiment was performed using the pre-incubation method in the presence of a reductive metabolic system. The test item was assayed at six dose levels from 156 to 5000 µg/plate. Neither precipitation of the test item, nor toxicity 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, in any experiment. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system.

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.