[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]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:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

EC Number:

265-449-9

EC Name:

[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

Cas Number:

65113-55-5

Molecular formula:

C25H30N3.C18H14N3O3S

IUPAC Name:

[4-[p,p'-bis(dimethylamino)benzhydrylidene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium m-[[p-anilinophenyl]azo]benzenesulphonate

Test material form:

solid: particulate/powder

Method

Target gene:

histidine operon and tryptophan operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1.2, 3.6, 12, 36, 120 µg/plate.
An additional concentration of 60 µg/plate was added in the second independent experiment for TA 1535 Salmonella typhimurium strain

Vehicle / solvent:

- solvent used: ethanol (60%)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

2-Nitrofluorene / sodium azide / 9-Aminoacridine / cis-Platinum (II) diammine dichloride / 2-Anthramine / Dimethyl-benzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar by direct incorporation or with pre-incubation for the second assay, if the first one is negative.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium):number of revertant colonies per plate

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 1-5 x 10 E 9

DETERMINATION OF CYTOTOXICITY
- Method: bacteriostatic activity - 3 assay

OTHER:
-Number of plates per assay: 3

Evaluation criteria:

R = (Number of revertant colonies wiith the test substance) / (Number of revertant colonies in the absence of the test substance)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

bacteriostatic activity of 75-76 % at the 120 µg/plate dose

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity: Bacteriostatic activity of 74-76% was observed with 120 µg/plate of test material. This bacteriostatic activity = the tolerated threshold of 75%.
No cytotoxic effect observed for the other doses.

COMPARISON WITH HISTORICAL CONTROL DATA:
Rate of spontaneous revertants and positive controls (with and without S9 mix) fall within the range of observed historical values at the facility.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For some strains with or without metabolic activation, a significative decrease of the number of revertants colonies is observed for the dose of 120 µg/plate.This finding is in accordance with the bacteriostatic activity observed for this concentration

Any other information on results incl. tables

STRAIN	DOSE/PLATE (µg)	R
		Assay 1: - S9 mix 10%	Assay 1: + S9 mix 10%	Assay 2: - S9 mix 10%	Assay 2: + S9 mix 10% and pre-incubation
TA 1535	120	0.20	0.18	0.35	0.93
	60	/	/	6.73	1.14
	36	3.53	0.93	4.73	0.93
	12	1.67	0.89	1.35	0.62
	3.6	0.93	1.36	0.77	0.62
	1.2	0.83	1.07	2.89	0.82
	Positive Control	55.03	13.31	62.09	4.89
TA 1537	120	0.35	0.31	0.45	0.79
	36	0.88	1.54	0.75	0.79
	12	1.53	1.12	1.45	0.93
	3.6	1.41	1.23	1.20	0.93
	1.2	1.18	1.19	0.15	0.90
	Positive Control	70.89	11.11	127.94	6.52
TA 98	120	0.22	0.27	0.36	1.36
	36	0.48	1.06	0.68	1.01
	12	0.89	1.00	0.96	1.12
	3.6	1.13	1.09	0.93	0.99
	1.2	1.15	1.15	1.00	0.94
	Positive Control	28.06	31.28	40.77	16.19
TA 100	120	0.24	0.39	0.33	0.54
	36	0.36	0.55	0.49	0.75
	12	0.52	0.95	0.70	0.92
	3.6	0.98	0.94	0.97	0.98
	1.2	1.05	1.01	0.90	0.96
	Positive Control	10.86	8.97	14.30	5.70
E.Coli	120	0.19	0.28	0.34	0.34
	36	0.43	1.14	0.54	1.26
	12	1.06	1.15	1.42	1.12
	3.6	0.86	1.12	1.11	1.19
	1.2	0.90	1.09	1.02	1.11
	Positive Control	6.87	5.44	13.44	4.66

R = (Number of revertant colonies in the presence of the test substance) / (Number of revertant colonies in the absence of the test substance)

Applicant's summary and conclusion

Conclusions:

positive without metabolic activation in TA 1535 Salmonella typhimurium

At 36 µg/plate for TA 1535 Salmonella typhimurium strain a significant increase in revertant number is observed without metabolic activation. Results were confirmed in a second independent experiment in which a higher dose of 60 µg/plate also induced a significant increase in revertant number.
There is no evidence of any increase in the number of revertant colonies in the presence of the test item (120, 36, 12, 3.6 and 1.2 µg/plate) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101). At 120 µg/plate for some strains a signiticant decrease in revertant number is observed with or without metabolic activation which is in accordance with the bacteriostatic activity observed for this concentration.

Executive summary:

The assessment of the potential mutagenic activity of the Substance was performed according to the Ames test (Salmonella His- and E.coli Trp- /microsome system) in compliance with the OECD Guideline 471 using the maximum tolerated concentration (standing below the threshold of 75%) recommended by OECD Guideline, i.e. 120 μg/plate for this toxicity assay. Four lower dilutions were chosen according to a geometrical (half-log) ratio were also tested.

 

Preparation of test material solution

The test material is diluted in ethanol 60%

 

Preliminary assay: Cytotoxicity

Five concentrations were studied. 0.1 mL of the bacterial suspension from a culture containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of the different concentrations of the test substance were successively added to 2 mL of overlay agar at 45°C, containing 10% (v/v) of a solution of L-Histidine-D-Biotine (2.5mM). After homogeneization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37°c, and the number of colonie counted.

A negative control containing the solvent alone was run in parallel.

 

Mutagenicity test

- Without metabolic activation:

Salmonella strains : for every strain, 0.1 mL of the bacterial suspension containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of every test substance concentration were successively added to 2 mL of overaly agar maintained surfusion in 45°C containing 10 % (v/v) of a L-Histidine-D-Biotin solution (0.5 mM).

 

E.Coli strain: in a test tube, 0.1 mL of the bacterial suspension containing 1 to 5 x 10⁹bacteria/mL and 0.1 mL of every test substance concentration were successively added to 2 mL of overaly agar maintained surfusion in 45°C containing 10 % (v/v) of Nutrient broth n°2 to which are added 5 µL of a L-Tryptophan solution at 2 mg/mL.

 

After homogeneization, the content of the tube was poured onto a Petri plate containing minimal agar (20 mL). 3 plates per concentration were incubated for 48 hours at 37°c, and the number of colonie counted.

 

- With metabolic activation :

Two techniques have been used:

- direct plate incorporation: Same technique to that described above, except that immediately before pouring the mixture onto the plates, 0.5 mL of S9-mix metabolic activation system is quickly mixed,

 

- by pre-incubation: The test substance is preincubated with the test strain, and 0.5 mL of S9 -mix metabolic activation system at least for 20 min at 37°C prior to mixing with the overlay agar and pouring onto the surface of the minimal agar plate.

 

If the first assay gives a positive response, the incorporation plate method has been performed for the second S9 mix assay.

If the first assay gives a negative response, the pre-incubation method has been performed for the the second S9 mix assay.

Solvent controls, positive controls were performed like in the mutagenicity assay without and with metabolic activation.

 

Bacterioastatic activity

Bacterioastatic activity: Cytotoxic rate of 76% and 75% was observed with 120 µg/plate of test material. This rate = the threshold of 75%. No cytotoxic effect observed for the other doses.

For all strains without metabolic activation and for TA 100- E.coli with metabolic activation, a significative decrease of the number of revertants colonies is observed for the dose of 120 µg/plate.This finding is in accordance with the bacteriostatic activity observed for this concentration.

 

Mutagenicity assays

The mutagenic activity of the test item was assessed by means of the Ames’s test in the four Salmonella typhimurium strains

TA1535, TA1537, TA98, TA100 and in the E. Coli WP2 (uvr A-) (pKM 101) tested either in presence or in absence of metabolic activation, in two independent assays. A mutagenic activity was found without metabolic activation in the TA 1535 Salmonella typhimurium strains at, in both experiments. Values fall within the range of historical values observed at the facility.

 

 

Conclusion

At 36 µg/plate for TA 1535 Salmonella typhimurium strain a significant increase in revertant number is observed without metabolic activation. Results were confirmed in a second independent experiment in which a higher dose of 60 µg/plate also induced a significant increase in revertant number.

There is no evidence of any increase in the number of revertant colonies in the presence of the test item (120, 36, 12, 3.6 and 1.2 µg/plate) without and with metabolic activation for bacterial strains in Salmonella typhimurium TA 1537, TA 98, TA 100 and Escherichia coli WP2(uvrA-) (pKM 101). At 120 µg/plate for some strains a significant decrease in revertant number is observed with or without metabolic activation which is in accordance with the bacteriostatic activity observed for this concentration.

 

In the assay conditions, the two concentrations of the test item 60 and 36 µg/plate induce reverse mutation on TA 1535 Salmonella typhimurium strain, without metabolic activation, according the OECD guidelines N°471 and to the method B13/B14 of the directive 2000/32/CE.