Bis[hydrogen [4-[4-(diethylamino)-5'-hydroxy-2',4'-disulphonatobenzhydrylidene]cyclohexa-2,5-dien-1-ylidene]diethylammonium], calcium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: Read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From December 07th to 19th, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July, 1997

Deviations:

yes

Remarks:

Only TA 98 strain is used

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:

Similar Substance 1

IUPAC Name:

Similar Substance 1

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

DOSE FIRST MAIN ASSAY -PLATE INCORPORATION METHOD:
TA98 (±S9): 5000, 2500, 1250, 625, 313 µg/plate
The concentration used in Main Assay I was chosen on the basis of the results obtained in the preliminary toxicity test. The test item was assayed at the maximum dose level of 5000 g/plate and at five lower dose levels spaced at approximately half-log intervals: 1580, 500, 158, 50.0 and 15.8 g/plate. No relevant toxicity was observed at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. A slight increase in revertant numbers was observed at the highest dose level tested in the presence of S9 metabolism. No relevant increase in revertant numbers was observed at any dose level in the absence of S9 metabolism. Since negative results were observed in Main Assay I, a pre-incubation step was included for treatment of Main Assay II.
DOSE SECOND MAIN ASSAY - PRE-INCUBATION METHOD:
TA98 (±S9): 5000, 2500, 1250, 625, 313 µg/plate
The dose-range of Main Assay II was slightly modified to take into account the results of Main Assay I.

Vehicle / solvent:

Solvent used: water.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix, (2 µg/plate) refers to the pre-incubation method assay.

Details on test system and experimental conditions:

TEST SYSTEM
Origin of strains: B. Ames Laboratory, University of California, Berkeley – USA
Storage: permanent stocks of this strain are kept at -80°C in RTC. 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 on Minimal plates + Biotin Growth on Minimal plates + Biotin + Histidine.
- uvrB: sensitivity to UV irradiation.
- rfa: sensitivity to Crystal Violet.
- pKM101: resistance to Ampicillin.

CULTURE MEDIA
- Nutrient Broth: Oxoid Nutrient Broth No 2 will be prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This will be used for the preparation of liquid cultures of the tester strain.
- Nutrient Agar: Oxoid Nutrient Broth No 2 (25g) and Difco Bacto-agar (l5g) will be added to one litre of distilled water and autoclaved. The solution will then be poured into plastic Petri dishes and allowed to solidify and dry before use. These plates will be used for the non-selective growth of the tester strain. lncubations on Nutrient Agar will be for approximately 48 or 72 hours.
- Minimal Agar: Minimal medium agar will be prepared as 1.5% Difco Bacto-agar in Voge1-Bonner Medium E, with 2% Glucose, autoclaved and poured into plastic Petri dishes.
- Top Agar: “Top Agar” (overlay agar) will be prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water. This solution will be autoclaved and stored. Prior to use 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine will be added to 100 ml of the top agar.

S9 TISSUE
One batch of S9 tissue fraction was used in this study and had the following characteristics:
- Species: Rat
- Strain Sprague: Sprague Dawley
- Tissue: Liver
- Inducing Agents: Phenobarbital – 5,6-Benzoflavone
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml): S9 tissue fraction 1.0 ml)+ NADP (100 mM) 0.4 ml+G-6-P (100 mM) 0.5 ml+ KCl (330 mM) 1.0 ml+ MgCl2 (100 mM) 0.8 ml+ Phosphate buffer (pH 7.4, 200 mM) 5.0 ml+ Distilled Water 1.3 ml

SOLUBILITY
The test item was found to be soluble in sterile distilled water at the concentration of 50.0 mg/ml. The solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. This result permitted a maximum concentration of 5000 g/plate to be used in the Main Assay I.

MAIN ASSAYS
Two 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 dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
- FIRST MAIN ASSAY -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. The overlay mixture was composed as follows: overlay agar (held at 45 °C) 2.0 ml +test or control item solution 0.1 ml+ S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml+ Bacterial suspension 0.1 ml.
- SECOND MAIN ASSAY- PRE-INCUBATION METHOD: the components were added in turn to an empty test-tube: bacterial suspension 0.1 ml+ test item solution or control item solution 0.05 ml+ S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml. 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, the scoring was affected by counting the number of revertant colonies on each plate.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
- Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
- The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
- No plates are lost through contamination or cracking.

Evaluation criteria:

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.

Statistics:

Scoring is effected by counting the number of revertant colonies on each plate, either manually, or using a Cardinal - Automatic colony counting system (Perceptive Instruments).

Results and discussion

Test results

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF PLATE INCORPORATION AND PRE-INCUBATION ASSAY.
In plate incorporation assay, no relevant toxicity was observed at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. A slight increase in revertant numbers was observed at the highest dose level tested in the presence of S9 metabolism. No relevant increase in revertant numbers was observed at any dose level in the absence of S9 metabolism.
Since negative results were observed in plate incorporation assay, a pre-incubation step was included. No toxicity was observed at any dose level in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. In the presence of S9 metabolic activation an increase of revertant numbers, almost reaching a two-fold increase over concurrent control, was observed at the highest dose level tested. In addition, the number of revertant colonies of this dose level was out of the range of historical negative control data. No increases in revertant numbers was observed in the absence 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.

ACCEPTANCE CRITERIA
All acceptance criteria were fulfilled.
- Results show that mean plate counts for untreated and positive control plates fell within the normal range (see attached tables).
- The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain.
-No plates were lost trough contamination or cracking.

EVALUATION OF THE RESULTS
Since the criteria for a clear positive result were not fully met, the test item could not be considered mutagenic.

Remarks on result:

other: Complementary to existing studies

Remarks:

Retest because of unclear results in previous studies

Applicant's summary and conclusion

Conclusions:

The test item does not induce reverse mutation in Salmonella Thiphimurium in the absence or presence of S9 metabolism.

Executive summary:

The test item PATENT BLUE V (E131)was examined for the ability to induce gene mutations in tester strain TA98 ofSalmonella typhimurium,as measured by reversion of auxotrophic strain to prototrophy. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item was used as a solution in sterile distilled water.

MAIN TESTS

- In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000mg/plate and at five lower dose levels spaced at approximately half-log intervals: 1580, 500, 158, 50.0 and 15.8mg/plate. No relevant toxicity was observed at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. A slight increase in revertant numbers was observed at the highest dose level tested in the presence of S9 metabolism. No relevant increase in revertant numbers was observed at any dose level in the absence of S9 metabolism.

- Since negative results were observed in Main Assay I, a pre-incubation step was included for treatment of Main Assay II. The test item was assayed at the maximum dose level of 5000mg/plate and at four lower dose levels spaced by a factor of two: 2500, 1250, 625 and 313mg/plate. No toxicity was observed at any dose level in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration. In the presence of S9 metabolic activation an increase of revertant numbers, (1.9 fold) over the concurrent negative control, was observed at the highest dose level tested. No increase in revertant numbers was observed in the absence of S9 metabolism.

CONCLUSIONS

It is concluded that the test item PATENT BLUE V (E131), for which the results obtained do not meet the criteria stated for positive results, is considered not mutagenic in this test. However, the slight but reproducible increases in revertant numbers observed only at the highest dose level, must be further investigated in additional genetoxicity assays.