Reaction mass of disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate and disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

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:

2018-02-15 till 2018-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

270-629-5

EC Name:

Disodium 4-amino-3-[[4-[(2-amino-4-hydroxyphenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68460-07-1

Molecular formula:

C28H22N8O8S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2-amino-4-hydroxyphenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Constituent 2

Reference substance name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

EC Number:

272-559-0

EC Name:

Disodium 4-amino-3-[[4-[(2,4-diaminophenyl)azo]phenyl]azo]-5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate

Cas Number:

68877-33-8

Molecular formula:

C28H23N9O7S2.2Na

IUPAC Name:

disodium 4-amino-3-({4-[(2,4-diaminophenyl)diazenyl]phenyl}diazenyl)-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Method

Species / strain

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

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Solvent used: DMSO
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

DURATION:
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:

positive

Remarks:

strain TA 98 with and without S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strain WP2 uvrA with and without S9 mix

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: Thick suspension

Other confounding effects: Due to the intense color of the test item the colonies were partly counted manually.

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2 uvrA with and without S9 mix at 5000 µg/plate. In the remaining strains no toxic effects were observed.

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1888600	Study Code: Envigo 1888600
Experiment: 1888600 VV Plate	Date Plated: 15.02.2018
Assay Conditions:	Date Counted: 22.02.2018

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	DMSO		10 ± 4	9 ± 3	26 ± 6	118 ± 6	36 ± 9
Activation	Untreated		9 ± 3	13 ± 4	25 ± 3	169 ± 21	33 ± 2
	Aluminium	3 µg	9 ± 3	9 ± 2	26 ± 6	119 ± 21	30 ± 4
	Black CRO	10 µg	12 ± 4	10 ± 4	22 ± 8	133 ± 5	32 ± 10
		33 µg	12 ± 5	8 ± 2	24 ± 6	131 ± 13	33 ± 9
		100 µg	10 ± 5	11 ± 1	23 ± 5	101 ± 4	32 ± 3
		333 µg	7 ± 3^D	11 ± 1^D	61 ± 6^D M	123 ± 5^D M	33 ± 11^D
		1000 µg	6 ± 1^M D	11 ± 5^M D	87 ± 5^M D	129 ± 12^M D	20 ± 8^M D
		2500 µg	6 ± 1^M D	13 ± 2^M D	125 ± 7^M D	126 ± 15^M D	20 ± 4^M D
		5000 µg	6 ± 3^M D	10 ± 1^M D	152 ± 14^M D	75 ± 15^M D	11 ± 4^M D
	NaN3	10 µg	1070 ± 73			1740 ± 120
	4-NOPD	10 µg			340 ± 9
	4-NOPD	50 µg		61 ± 7
	MMS	2.0 µL					742 ± 59
With	DMSO		12 ± 3	13 ± 5	37 ± 1	109 ± 2	42 ± 6
Activation	Untreated		16 ± 3	13 ± 2	48 ± 9	146 ± 13	44 ± 1
	Aluminium	3 µg	14 ± 0	11 ± 1	46 ± 3	112 ± 4	35 ± 8
	Black CRO	10 µg	9 ± 2	13 ± 3	64 ± 3	119 ± 8	42 ± 1
		33 µg	8 ± 3	18 ± 2	94 ± 2	135 ± 12	36 ± 6
		100 µg	11 ± 4	19 ± 3	151 ± 30	133 ± 11	36 ± 7
		333 µg	7 ± 2^D	14 ± 2^D	202 ± 9^D M	114 ± 12^D M	31 ± 5^D
		1000 µg	8 ± 2^M D	15 ± 2^M D	86 ± 15^M D	125 ± 16^M D	27 ± 3^M D
		2500 µg	8 ± 3^M D	13 ± 2^M D	119 ± 8^M D	120 ± 18^M D	26 ± 2^M D
		5000 µg	6 ± 1^M D	9 ± 2^M D	131 ± 13^M D	125 ± 9^M D	12 ± 4^M D
	2-AA	2.5 µg	355 ± 9	220 ± 14	3739 ± 231	2586 ± 60
	2-AA	10.0 µg					467 ± 36

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

Applicant's summary and conclusion

Conclusions:

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 with and without metabolic activation (S9 mix).

Executive summary:

This study was performed to investigate the potential of Aluminium Black CRO to induce gene mutations according to the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 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.

Toxiceffects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in strain WP2uvrAwith and without S9 mix at 5000µg/plate. In the remaining strains no toxic effects were observed.

A substantial increase in revertant colony numbers was observed following treatment with Aluminium Black CRO in strain TA 98 in the presence and absence of metabolic activation (S9 mix).The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations of 333 µg/plate and above without S9 mix and at concentrations of 33 µg/plate and above with S9 mix. In strain TA 98 with S9 mix the revertant colony count decreases at concentrations of 1000 µg/plate and above, but the values did not fall below the threshold of twice the number of the corresponding solvent control.

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