Reaction products of diazotised 2-amino-5-{[2-(sulfooxy)ethyl]sulfonyl}benzenesulfonic acid coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid under acidic conditions, further coupled with diazotised reaction products of 2,4,6-trifluoro-1,3,5-triazine with 2-[(2-anilinoethyl)sulfonyl]ethyl hydrogen sulfate and 2,4-diaminobenzenesulfonic acid (1:1:1) under alkaline conditions, potassium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in the OECD 471 bacterial reverse mutation assay with Prival modification with and without metabolic activation.

Link to relevant study records

Reference

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:

2018-10-02 till 2018-10-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

guideline-conform study under GLP without deviations

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

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9 (experiment I) non-induced hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

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: 30 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 of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above 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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

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, solvent
Precipitation: No precipitation of the test item occurred up to the highest investigated dose, but an intense color in the overlay agar of the plates incubated with the test item was observed from 2500 to 5000 µg/plate in both experiments.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, In experiment II, the data in solvent control of strain TA 98 with S9 mix were slightly above our historical control range (60 versus 56 colonies). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no detrimental impact on the outcome of the study.
ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Summary of Experiment I

Study Name: 1922401	Study Code: Envigo 1922401
Experiment: 1922401 VV Plate	Date Plated: 02.10.2018
Assay Conditions:	Date Counted: 05.10.2018

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Deionised water		11 ± 4	11 ± 4	34 ± 3	114 ± 19	43 ± 5
Activation	Untreated		7 ± 3	9 ± 2	35 ± 9	135 ± 9	47 ± 7
	Reactive	3 µg	12 ± 4	11 ± 3	34 ± 8	114 ± 14	42 ± 7
	Blue	10 µg	13 ± 3	10 ± 3	29 ± 3	115 ± 3	40 ± 10
	F07-0195	33 µg	9 ± 3	12 ± 5	35 ± 4	126 ± 7	45 ± 6
		100 µg	12 ± 3	10 ± 3	27 ± 5	128 ± 23	42 ± 7
		333 µg	12 ± 4	11 ± 5	28 ± 9	131 ± 15	44 ± 2
		1000 µg	9 ± 1	7 ± 2	27 ± 6	112 ± 16	35 ± 7
		2500 µg	9 ± 3^D M	8 ± 2^D M	19 ± 1^D M	107 ± 6^D M	45 ± 4^D M
		5000 µg	8 ± 1^D M	8 ± 2^D M	16 ± 3^D M	98 ± 8^D M	52 ± 6^D M
	NaN3	10 µg	1101 ± 46			1699 ± 102
	4-NOPD	10 µg			508 ± 14
	4-NOPD	50 µg		91 ± 4
	MMS	2.0 µL					939 ± 54
With	Deionised water		15 ± 6	11 ± 4	33 ± 3	154 ± 3	53 ± 17
Activation	Untreated		10 ± 4	15 ± 4	40 ± 3	150 ± 6	51 ± 3
	Reactive	3 µg	13 ± 6	16 ± 6	38 ± 6	139 ± 38	54 ± 7
	Blue	10 µg	16 ± 1	11 ± 2	46 ± 5	151 ± 22	52 ± 5
	F07-0195	33 µg	15 ± 4	18 ± 2	35 ± 9	144 ± 7	52 ± 3
		100 µg	14 ± 4	17 ± 4	38 ± 5	146 ± 5	54 ± 2
		333 µg	12 ± 5	16 ± 2	37 ± 11	144 ± 9	51 ± 5
		1000 µg	15 ± 3	11 ± 5	31 ± 5	128 ± 12	38 ± 11
		2500 µg	10 ± 3^D M	6 ± 2^D M	20 ± 2^D M	96 ± 6^D M	44 ± 3^D M
		5000 µg	7 ± 1^D M	5 ± 2^D M	17 ± 4^D M	94 ± 5^D M	45 ± 9^D M
	2-AA	2.5 µg	445 ± 25	188 ± 22	3579 ± 273	4001 ± 23
	2-AA	10.0 µg					347 ± 16

Summary of Experiment II

Study Name: 1922401	Study Code: Envigo 1922401
Experiment: 1922401 HV2 Pre	Date Plated: 10.10.2018
Assay Conditions:	Date Counted: 17.10.2018

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	Deionised water		12 ± 4	9 ± 1	30 ± 5	118 ± 24	52 ± 9
Activation	Untreated		15 ± 5	14 ± 7	33 ± 4	128 ± 7	48 ± 3
	Reactive	33 µg	15 ± 5	11 ± 1	36 ± 1	113 ± 3	47 ± 10
	Blue	100 µg	13 ± 2	12 ± 4	43 ± 9	136 ± 10	58 ± 6
	F07-0195	333 µg	12 ± 3	12 ± 2	30 ± 4	152 ± 18	57 ± 8
		1000 µg	12 ± 3	9 ± 3	28 ± 9	135 ± 13	52 ± 9
		2500 µg	9 ± 2^D M	8 ± 3^D M	23 ± 5^D M	109 ± 11^D M	47 ± 7^D M
		5000 µg	9 ± 2^D M	6 ± 1^D M	17 ± 7^D M	107 ± 24^D M	56 ± 9^D M
	NaN3	10 µg	1229 ± 54			1810 ± 26
	4-NOPD	10 µg			571 ± 44
	4-NOPD	50 µg		87 ± 8
	MMS	2 µL					1054 ± 46
With	Deionised water		16 ± 4	22 ± 3	60 ± 6	156 ± 11	48 ± 8
Activation	Untreated		16 ± 4	19 ± 5	47 ± 1	143 ± 15	53 ± 6
	Reactive	33 µg	19 ± 1	28 ± 5	62 ± 6	156 ± 12	55 ± 5
	Blue	100 µg	14 ± 3	27 ± 3	62 ± 9	152 ± 19	42 ± 6
	F07-0195	333 µg	17 ± 5	24 ± 4	46 ± 12	150 ± 6	45 ± 4
		1000 µg	13 ± 3	21 ± 5	41 ± 5	171 ± 6	55 ± 4
		2500 µg	15 ± 2^D M	18 ± 1^D M	37 ± 6^D M	138 ± 15^D M	47 ± 7^D M
		5000 µg	12 ± 3^D M	14 ± 3^D M	27 ± 9^D M	99 ± 4^D M	53 ± 6^D M
	2-AA	2.5 µg				4276 ± 423
	2-AA	2.5 µg	333 ± 11	528 ± 9
	2-AA	10 µg					976 ± 47
	Congo red	500 µg			252 ± 51

Key to Plate Postfix Codes:              

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

Congo Red

Conclusions:

In conclusion, it can be stated that 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:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration, including the controls, was 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:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose, but an intense color in the overlay agar of the plates incubated with the test item was observed from 2500 to 5000 µg/plate in both experiments.

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 all strains with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with the test item at any dose 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 and showed a distinct increase of induced revertant colonies.

In experiment II, the data in solvent control of strain TA 98 with S9 mix were slightly above our historical control range (60 versus 56 colonies). Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies and has no detrimental impact on the outcome of the study.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was not found to induce gene mutation in bacteria in an OECD 471 guideline study with and without metabolic activation. No classification applies.