Sulfonation products of (N-phenyl-4-[[4-(phenylamino)phenyl][4-(phenylimino)cyclohexa-2,5-dien-1-ylidene]methyl]aniline, monohydrochloride and 4,4'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]bis[N-phenylaniline], monohydrochloride), sodium salts

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:

2017-01-17 till 2017-02-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

EC Number:

944-086-6

IUPAC Name:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

Test material form:

solid

Details on test material:

UVCB

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent used: deionised water
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 60 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

Remarks on result:

other:

Remarks:

the test item did not induce gene mutations

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1802902	Study Code: Envigo 1802902
Experiment: 1802902 VV Plate	Date Plated: 17.01.2017
Assay Conditions:	Date Counted: 20.01.2017

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without	A. deion.		13 ± 6	10 ± 3	24 ± 3	135 ± 7	44 ± 3
Activation	Untreated		15 ± 4	9 ± 2	28 ± 2	144 ± 9	37 ± 6
	Test item	3 µg	12 ± 6	9 ± 3	27 ± 2	137 ± 11	35 ± 7
		10 µg	12 ± 4	9 ± 0	27 ± 6	116 ± 5	34 ± 8
		33 µg	11 ± 1	9 ± 1	23 ± 7	143 ± 13	39 ± 6
		100 µg	9 ± 5	10 ± 1	23 ± 7	141 ± 23	35 ± 5
		333 µg	10 ± 2	8 ± 2	27 ± 5	132 ± 23	44 ± 3
		1000 µg	13 ± 1	8 ± 2	21 ± 2	115 ± 10^D M	42 ± 2
		2500 µg	15 ± 3^D M	8 ± 2^D M	21 ± 2^D M	109 ± 11^D M	34 ± 4^D M
		5000 µg	7 ± 1^D M P	6 ± 1^D M P	21 ± 2^D M P	102 ± 10^D M P	34 ± 4^D M P
	NaN3	10 µg	1346 ± 108			2133 ± 98
	4-NOPD	10 µg			424 ± 31
	4-NOPD	50 µg		80 ± 5
	MMS	2.0 µL					978 ± 38
With	A. deion.		11 ± 6	10 ± 4	32 ± 7	122 ± 12	47 ± 6
Activation	Untreated		12 ± 5	11 ± 3	45 ± 4	123 ± 5	49 ± 8
	Test item	3 µg	12 ± 6	10 ± 1	39 ± 8	122 ± 7	47 ± 5
		10 µg	11 ± 5	12 ± 3	32 ± 5	119 ± 6	53 ± 5
		33 µg	11 ± 1	10 ± 3	32 ± 7	117 ± 12	51 ± 5
		100 µg	9 ± 2	13 ± 2	40 ± 5	124 ± 6	53 ± 11
		333 µg	12 ± 2	11 ± 4	45 ± 3	121 ± 9	42 ± 9
		1000 µg	14 ± 6	11 ± 1	42 ± 8	131 ± 6	44 ± 5
		2500 µg	11 ± 1	12 ± 3	37 ± 3	114 ± 14^D M	49 ± 9
		5000 µg	13 ± 3^D M P	9 ± 1^D M P	25 ± 3^D M P	113 ± 9^D M P	43 ± 7^D M P
	2-AA	2.5 µg	469 ± 21	120 ± 13	4399 ± 760	4016 ± 110
	2-AA	10.0 µg					357 ± 48

Summary of Experiment II

Study Name: 1802902	Study Code: Envigo 1802902
Experiment: 1802902 HV2 Pre	Date Plated: 25.01.2017
Assay Conditions:	Date Counted: 01.02.2017

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without	A. deion.		13 ± 4	10 ± 4	29 ± 6	209 ± 1	34 ± 3
Activation	Untreated		11 ± 4	10 ± 5	28 ± 4	204 ± 9	39 ± 4
	Test item	10 µg	12 ± 3	9 ± 4	26 ± 5	214 ± 18	39 ± 4
		33 µg	15 ± 4	10 ± 0	24 ± 2	192 ± 13	36 ± 3
		100 µg	16 ± 5	10 ± 3	27 ± 3	178 ± 39	41 ± 7
		333 µg	16 ± 1	10 ± 3	26 ± 5	149 ± 16	33 ± 12
		1000 µg	11 ± 1^D	11 ± 4^D	23 ± 8^D	187 ± 8^M D	28 ± 8^D
		2500 µg	11 ± 2^M D	9 ± 4^M D	27 ± 3^M D	181 ± 12^M D	27 ± 8^M D
		5000 µg	11 ± 4^P M D	8 ± 2^P M D	23 ± 5^P M D	175 ± 19^P M D	25 ± 1^P M D
	NaN3	10 µg	1355 ± 101			1873 ± 158
	4-NOPD	10 µg			525 ± 34
	4-NOPD	50 µg		99 ± 7
	MMS	2.0 µL					742 ± 32
With	A. deion.		12 ± 5	11 ± 2	33 ± 3	198 ± 25	46 ± 6
Activation	Untreated		12 ± 5	9 ± 4	41 ± 11	193 ± 29	41 ± 6
	Test item	10 µg	14 ± 4	11 ± 1	44 ± 6	206 ± 3	48 ± 6
		33 µg	10 ± 3	13 ± 3	29 ± 2	198 ± 14	50 ± 3
		100 µg	11 ± 1	9 ± 4	39 ± 12	182 ± 3	48 ± 9
		333 µg	15 ± 5	10 ± 5	51 ± 9	206 ± 19	48 ± 6
		1000 µg	15 ± 6^D	12 ± 2^D	35 ± 8^D	189 ± 22^D	47 ± 8^D
		2500 µg	17 ± 7^D	10 ± 6^D	31 ± 5^D	189 ± 7^M D	36 ± 10^D
		5000 µg	10 ± 3^P M D	8 ± 2^P M D	24 ± 5^P M D	190 ± 24^P M D	35 ± 3^P M D
	2-AA	2.5 µg	374 ± 18	137 ± 16	3336 ± 515	3451 ± 343
	2-AA	10.0 µg					347 ± 8

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

D:  Densely Colored Plate

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                            10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 µg/plate. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.