Hydrogen [4-[[4-(diethylamino)phenyl][4-[ethyl[(3-sulphonatobenzyl)amino]-o-tolyl]methylene]-3-methylcyclohexa-2,5-dien-1-ylidene](ethyl)(3-sulphonatobenzyl)ammonium, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test according to OECD TG 471 ( Bacterial Reverse Mutation Assay) Special Brilliant Blue FFR [CAS no. 6505-30-2] induced gene mutations by frameshifts in the genome of strain TA 98 in the presence of S9 mix. No indications for gene mutations were observed for the other Salmonella typhimurium strains or for Escherichia coli in this reverse mutation assay.

Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) a combined micronucleus assay and alkaline comet assay in the rat is proposed to evaluate potential mutagenic activity in vivo.

The tests shall be conducted according to OECD TG 489 (in vivo mammalian alkaline comet assay) combined with OECD TG 474 (Mammalian erythrocyte micronucleus test) on rats by the oral route (gavage). The following systemic and local organs shall be analysed: liver, glandular stomach, duodenum/ jejunum and bone marrow. For the micronucleus test the bone marrow shall be analysed (see IUCLID section 7.6.2).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 18 December 2017 Experimental completion date: 24 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physical State / Appearance: Solid, crystalline, brown shiny metallic
Expiry Date: 22 June 2018
Storage Conditions: At room temperature
Stability in Solvent: Stable in DMSO for 4 and 24 hours*
* Data generated in a separate GLP study (Envigo Ref No: LF04LF) conducted at Envigo Research Ltd.
The dose selection was adjusted to purity.

Target gene:

histidine locus in S. typhimurium and trptophan locus in E. coli.

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:

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 Ia:
Strain TA 98 with S9 mix: 100; 250; 500; 750; 1000; 1500; 2500; and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item Special Brilliant Blue FFR was dissolved in DMSO (purity > 99 %). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA 1535, TA 100

Positive control substance:

sodium azide

Remarks:

Absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate in strain TA 98, 50 µg/plate in strain TA 1537

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2.0 µL/plate for WP2 uvrA

Positive control substance:

methylmethanesulfonate

Remarks:

Absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate for all strains except WP2 uvrA, which was treated at 10.0 µg/plate

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria were met:
Evaluable plates (>0 colonies) at five concentrations or more were used in all strains.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since mutagenic effects were observed in experiment I, a confirmatory experiment Ia was performed as a plate incorporation assay. 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment Ia:
100; 250; 500; 750; 1000; 1500; 2500; and 5000 µg/plate

Experimental Performance
For each strain and dose level, including the controls, three plates were used.
Plate Incorporation Assay
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µl S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Data Evaluation
Data Recording
In experiment I the colonies were counted using a validated computer system (cf. 3.8, Major computerized systems), which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to the intense color of the test item the colonies were partly counted manually.
In experiment Ia the colonies were counted manually due to a failure of the computer system.
 

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above (strains TA 98, TA 100, and WP2 uvrA) or of thrice 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.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effects without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

minor toxic effects

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item Special Brilliant Blue FFR 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 typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain 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 Ia:
Strain TA 98 with S9 mix: 100; 250; 500; 750; 1000; 1500; 2500; and 5000 µg/plate
No precipitation of the test item occurred up to the highest investigated dose.
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.
Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strains TA 1537 and TA 98 without metabolic activation at 5000 µg/plate.
In experiment I an increase in revertant colony numbers reaching or exceeding the threshold of twice the number in revertant colony count of the corresponding solvent control, was observed following treatment with Special Brilliant Blue in strain TA 98 in the presence of metabolic activation (S9 mix). The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at 1000 µg/plate. At the next higher concentrations the number of colonies decreased to an induction factor of 0.5 at 5000 µg/plate.
To verify the positive results in experiment I a confirmatory experiment Ia was performed as a plate incorporation assay with strain TA 98 with S9 mix. In experiment Ia the positive result of experiment I was confirmed. The threshold of twice the number of the corresponding solvent control was reached at 250 µg/plate and exceeded at concentrations from 500 to 1000 µg/plate. At the higher concentrations the number in revertant colonies decreased below the threshold of twice the number of the corresponding solvent control.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Summary of Experiment I

Study Name: 1856001	Study Code: Envigo 1856001
Experiment: 1856001 VV Plate	Date Plated: 18.12.2017
Assay Conditions:	Date Counted: 21.12.2017

 

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 2	11 ± 2	24 ± 4	129 ± 8	33 ± 7
	Untreated		11 ± 3	13 ± 2	17 ± 6	129 ± 8	32 ± 4
	Special	3 µg	7 ± 0	9 ± 3	23 ± 8	135 ± 16	25 ± 4
	Brilliant	10 µg	10 ± 4	9 ± 4	19 ± 5	139 ± 7	28 ± 9
	Blue FFR	33 µg	10 ± 1	11 ± 1	24 ± 6	128 ± 10	27 ± 5
		100 µg	10 ± 2	9 ± 5	25 ± 8	139 ± 17	30 ± 9
		333 µg	10 ± 3^D M	8 ± 3^D M	16 ± 2^D M	133 ± 8^D M	22 ± 4^D M
		1000 µg	8 ± 3^D M	7 ± 2^D M	16 ± 2^D M	129 ± 17^D M	21 ± 5^D M
		2500 µg	8 ± 3^D M	5 ± 2^D M	15 ± 4^D M	121 ± 18^D M	18 ± 3^D M
		5000 µg	5 ± 2^D M	3 ± 2^D M	10 ± 3^D M	83 ± 6^D M	16 ± 3^D M
	NaN3	10 µg	1121 ± 26			1971 ± 236
	4-NOPD	10 µg			310 ± 6
	4-NOPD	50 µg		142 ± 24
	MMS	2.0 µL					873 ± 35
With Activation	DMSO		12 ± 3	12 ± 4	26 ± 6	118 ± 21	39 ± 7
	Untreated		13 ± 3	9 ± 4	37 ± 10	135 ± 10	47 ± 5
	Special	3 µg	13 ± 3	10 ± 1	32 ± 4	116 ± 9	31 ± 9
	Brilliant	10 µg	13 ± 6	8 ± 3	24 ± 3	123 ± 5	32 ± 6
	Blue FFR	33 µg	12 ± 5	10 ± 2	33 ± 5	120 ± 14	30 ± 9
		100 µg	12 ± 5	8 ± 2	28 ± 3	109 ± 6	32 ± 10
		333 µg	10 ± 2^D M	11 ± 2^D M	48 ± 4^D M	119 ± 16^D M	27 ± 1^D M
		1000 µg	9 ± 1^D M	10 ± 2^D M	67 ± 10^D M	115 ± 11^D M	26 ± 2^D M
		2500 µg	8 ± 2^D M	8 ± 1^D M	54 ± 7^D M	101 ± 6^D M	23 ± 2^D M
		5000 µg	7 ± 1^D M	7 ± 1^D M	14 ± 3^D M	92 ± 5^D M	19 ± 2^D M
	2-AA	2.5 µg	430 ± 12	113 ± 11	3688 ± 304	4084 ± 196
	2-AA	10.0 µg					331 ± 19

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	D M	Densely coloured plate Manual count

 

Summary of Experiment Ia

Study Name: 1856001	Study Code: Envigo 1856001
Experiment: 1856001 VVa Plate	Date Plated: 18.01.2018
Assay Conditions:	Date Counted: 24.01.2018

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 98
With Activation	DMSO			34 ± 1
	Untreated			30 ± 0
	Special	100µg		40 ± 10
	Brilliant	250µg		68 ± 4
	Blue FFR	500µg		85 ± 9
		750µg		98 ± 11
		1000µg		69 ± 8
		1500 µg		54 ± 6
		2500 µg		49 ± 7
		5000 µg		28 ± 6
	2-AA	2.5 µg		3050 ± 95

 

Key to Positive Controls
2-AA	2-aminoanthracene

 

 

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 in the presence of S9 mix.

Executive summary:

Summary

This study was performed to investigate the potential of Special Brilliant Blue FFR to induce gene mutations according to the plate incorporation test (experiment I and IIa) using theSalmonella 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, 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 Ia:

Strain TA 98 with S9 mix:              100; 250; 500; 750; 1000; 1500; 2500; and 5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose.

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.

Minor toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment I in strains TA 1537 and TA 98 without metabolic activation at 5000 µg/plate.

A substantial increase in revertant colony numbers was observed following treatment with Special Brilliant Blue FFR in strain TA 98 in the presence of S9 mix in experiment I and Ia.

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

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 in the presence of S9 mix.

Therefore, Special Brilliant Blue FFR is considered to be mutagenic in Salmonella typhimurium TA 98; no indications for gene mutations were observed for the other Salmonella typhimurium strains or for Escherichia coli in this reverse mutation assay

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No genotoxicity studies in vivo studies are available.

Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) a combined micronucleus assay and alkaline comet assay in the rat is proposed to evaluate potential mutagenic activity in vivo.

The tests shall be conducted according to OECD TG 489 (in vivo mammalian alkaline comet assay) combined with OECD TG 474 (Mammalian erythrocyte micronucleus test) on rats by the oral route (gavage). The following systemic and local organs shall be analysed: liver, glandular stomach, duodenum/ jejunum and bone marrow. For the micronucleus test the bone marrow shall be analysed (see IUCLID section 7.6.2).

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In an Ames test according to OECD TG 471 (Bacterial Reverse Mutation Assay) Special Brilliant Blue FFR [CAS no. 6505-30-2] induced gene mutations by frameshifts in the genome of strain TA 98 in the presence of S9 mix. No indications for gene mutations were observed for the other Salmonella typhimurium strains or for Escherichia coli in this reverse mutation assay.

Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) a combined micronucleus assay and alkaline comet assay in the rat is proposed to evaluate potential mutagenic activity in vivo.

The tests shall be conducted according to OECD TG 489 (in vivo mammalian alkaline comet assay) combined with OECD TG 474 (Mammalian erythrocyte micronucleus test) on rats by the oral route (gavage). The following systemic and local organs shall be analysed: liver, glandular stomach, duodenum/ jejunum and bone marrow. For the micronucleus test the bone marrow shall be analysed (see IUCLID section 7.6.2).

A classification for genotoxicity of Special Brilliant Blue FFR [CAS no. 6505-30-2] is postponed until the results of the combined micronucleus assay and alkaline comet assay in the rat are available.