Heptasodium bis[4-hydroxy-5-[(2-hydroxy-1-naphthyl)azo]-3-[(2-hydroxy-3-nitro-5-sulphophenyl)azo]naphthalene-2,7-disulphonato(5-)]cobaltate(7-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 23. Oct. to 03. Dec. 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read-across justification is detailed in section 13; source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

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:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid

Method

Target gene:

HIS /TRP

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix (rat and hamster)

Test concentrations with justification for top dose:

- 33, 100, 333, 1000, 2600 and 5200 μg/plate (SPT)
- 33, 100, 333, 1000, 2600 and 5200 μg/plate (Prival test)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

treated with soft agar, S9 mix, buffer, vehicle or the test item without the addition of tester strains

Negative solvent / vehicle controls:

yes

Remarks:

contains vehicle used at the same concentration and volume (all strains)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With rat liver S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

treated with soft agar, S9 mix, buffer, vehicle or the test item without the addition of tester strains

Negative solvent / vehicle controls:

yes

Remarks:

contains vehicle used at the same concentration and volume (all strains)

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With hamster liver S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

treated with soft agar, S9 mix, buffer, vehicle or the test item without the addition of tester strains

Negative solvent / vehicle controls:

yes

Remarks:

contains vehicle used at the same concentration and volume (all strains)

Positive controls:

yes

Positive control substance:

9-aminoacridine

other:

not specified

Remarks:

Without S9 mix

Details on test system and experimental conditions:

STANDARD PLATE TEST
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.

Salmonella typhimurium
Test tubes containing 2 ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % w/v agar + 0.6% w/v NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45 °C, and the remaining components were added in the following order:
- 0.1 ml test solution or vehicle (negative control)
- 0.1 ml fresh bacterial culture
- 0.5 ml S9 mix (with metabolic activation) or 0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds and incubated at 37 °C for 48 to 72 hours in the dark, and the bacterial colonies (his+ revertants) were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2 ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8 % w/v agar + 0.6% w/v NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
- 0.1 ml test solution or vehicle (negative control)
- 0.1 ml fresh bacterial culture
- 0.5 ml S9 mix (with metabolic activation) or 0.5 ml phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37 °C for 48 to 72 hours in the dark, the bacterial colonies (his+ revertants) were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

PRIVAL PREINCUBATION TEST
This experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. and has been modified further to include reductive conditions by Prival et al.
0.1 ml test solution or vehicle (negative control), 0.1 ml bacterial suspension and 0.5 ml S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 30 °C for 30 minutes using a shaker. Subsequently, 2 ml soft agar which consists of 100 ml agar (0.8% w/v agar + 0.6% w/v NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin or 0.5 mM tryptophan) were added. After mixing, the samples were poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition of the minimal glucose agar:
- 980 ml purified water
- 20 ml Vogel-Bonner E medium
- 15 g Difco bacto agar
- 5 g D-glucose, monohydrate
After incubation at 37 °C for 48 to 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
- the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain;
- the sterility controls revealed no indication of bacterial contamination;
- the positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above;
- fresh bacterial culture containing approximately 10^9 cells per ml were used.

Assessment criteria
The test item was considered positive in this assay if the following criteria were met: a dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test item was generally considered non-mutagenic in this test if: the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward.
In the prival preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward.

SOLUBILITY
Test substance precipitation was found from about 2600 μg/plate onward with and without S9 mix.

CONTROL
The number of revertant colonies in the negative controls was within or marginally below the range of the historical negative control data for each tester strain, with or without metabolic activation.
The positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Any other information on results incl. tables

Table 1: Results of the standard plate test without metabolic activation.

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	SD	Factor	Individual revertant colony counts
TA 1535	DMSO	-	14.7	3.1	-	14, 12, 18
	test item	33	11.7	1.5	0.8	13, 12, 10
		100	11.3	6.0	0.8	17, 12, 5
		333	12.7	5.8	0.9	16, 16, 6
		1000	6.7	2.1	0.5	9, 6, 5
		2600	6.7	4.0	0.5	3 P, 6 P, 11 P
		5200	5.0	3.0	0.3	2 P, 5 P, 8 P
	MNNG	5.0	4180.7	120.7	285	4049, 4286, 4207
TA 100	DMSO	-	45	6.2	-	38, 50, 47
	test item	33	57.3	5.0	1.3	58, 62, 52
		100	61.0	5.2	1.4	58, 58, 67
		333	53.3	13.0	1.2	66, 40, 54
		1000	51.7	4.0	1.1	48, 51, 56
		2600	53.0	7.8	1.2	48 P, 62 P, 49 P
		5200	45.0	7.0	1.0	42 P, 40 P, 53 P
	MNNG	5.0	3017.7	134	67.1	2999, 2894, 3160
TA 1537	DMSO	-	8.0	1.7	-	10, 7, 7
	test item	33	7.3	1.5	0.9	6, 7, 9
		100	8.0	5.0	1.0	13, 3, 8
		333	7.0	3.5	0.9	3, 9, 9
		1000	6.3	2.3	0.8	5, 5, 9
		2600	7.7	2.9	1.0	6 P, 6 P, 11 P
		5200	6.7	4.7	0.8	3 P, 12 P, 5 P
	AAC	100.0	794.3	177.1	99.3	859, 930, 594
TA 98	DMSO	-	27.0	2.6	-	30, 26, 25
	test item	33	27.7	3.8	1.0	25, 32, 26
		100	24.7	4.0	0.9	27, 20, 27
		333	25.7	4.0	1.0	30, 25, 22
		1000	19.7	1.5	0.7	20, 18, 21
		2600	13.3	4.9	0.5	11 P, 10 P, 19 P
		5200	10.3	5.7	0.4	12 P, 15 P, 4 P
	NOPD	10.0	360.3	31.0	13.3	373, 383, 325
E. coli	DMSO	-	48.7	9.8	-	43, 43, 60
	test item	33	50.0	4.6	1.0	45, 51, 54
		100	58.0	10.4	1.2	52, 52, 70
		333	58.7	4.9	1.2	62, 53, 61
		1000	46.3	5.9	1.0	42, 53, 44
		2600	45.0	19.0	0.9	40 P, 66 P, 29 P
		5200	35.3	12.4	0.7	21 P, 42 P, 43 P
	4-NQO	5.0	1030.3	5.1	21.2	1026, 1029, 1036

DMSO = dimethyl sulfoxide

MMNG = N-methyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate in DMSO

AAC = 9-aminoacradine 100 µg/plate in DMSO

NOPD = 4-nitro-o-phenylenediamine: 10 µg/plate in DMSO

4-NQO = 4-nitroquinoline-N-oxide 5 μg/plate in DMSO

SD = standard deviation

Table 2: Results of the standard plate test with metabolic activation.

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	SD	Factor	Individual revertant colony counts
TA 1535	DMSO	-	9.7	2.1	-	9, 12, 8
	test item	33	12.0	1.7	1.2	11, 14, 11
		100	10.7	0.6	1.1	11, 11, 10
		333	9.7	3.2	1.0	12, 11, 6
		1000	8.7	4.0	0.9	11, 4, 11
		2600	8.0	1.0	0.8	9 P, 7 P, 8 P
		5200	3.7	2.1	0.4	6 P, 3 P, 2 P
	2-AA	2.5	188.0	21	19.4	167, 209, 188
TA 100	DMSO	-	57.0	7.0	-	49, 62, 60
	test item	33	58.7	5.9	1.0	61, 63, 52
		100	69.3	8.0	1.2	77, 61, 70
		333	63.0	5.2	1.1	69, 60, 60
		1000	43.7	5.1	0.8	48, 38, 45
		2600	55.3	2.9	1.0	57 P, 52 P, 57 P
		5200	50.3	2.1	0.9	51 P, 48 P, 52 P
	2-AA	2.5	1815.0	420.0	31.8	2084, 2030, 1331
TA 1537	DMSO	-	8.0	3.5	-	10, 10, 4
	test item	33	7.0	1	0.9	6, 7, 8
		100	5.3	1.2	0.7	6, 6, 4
		333	6.0	1.7	0.8	4, 7, 7
		1000	7.3	2.9	0.9	9, 4, 9
		2600	5.3	1.5	0.7	7 P, 4 P, 5 P
		5200	6.0	1.0	0.8	5 P, 6 P, 7 P
	2-AA	2.5	154.7	32.3	19.3	192, 137, 135
TA 98	DMSO	-	26.3	2.3	-	25, 29, 25
	test item	33	24.0	6.6	0.9	31, 23, 18
		100	23.3	2.3	0.9	22, 22, 26
		333	20.3	3.1	0.8	23, 17, 21
		1000	21.7	4.5	0.8	20, 18, 21
		2600	17.0	10.0	0.6	27 P, 7 P, 17 P
		5200	12.7	5.9	0.5	6 P, 17 P, 15 P
	2-AA	2.5	1577.3	252.4	59.9	1286, 1716, 1730
E. coli	DMSO	-	59.7	11.4	-	47, 63, 69
	test item	33	57.3	4.7	1.0	61, 52, 59
		100	55.3	9.1	0.9	47, 65, 54
		333	56.0	1.7	0.9	58, 55, 55
		1000	54.0	2.6	0.9	56, 51, 55
		2600	51.7	1.5	0.9	50 P, 53 P, 52 P
		5200	33.7	2.1	0.6	33 P, 32 P, 36 P
	2-AA	60.0	228.0	37.5	3.8	265, 190, 229

DMSO = dimethyl sulfoxide

SD = standard devation

2-AA = 2-aminoanthracene

Table 3: Results of the Prival preincubation test without metabolic activation.

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	SD	Factor	Individual revertant colony counts
TA 1535	DMSO	-	13.3	2.1	-	15, 14, 11
	test item	33	15.7	4.2	1.2	19, 17, 11
		100	10.3	4.0	0.8	14, 11, 6
		333	12.7	2.5	1.0	13, 15, 10
		1000	11.7	0.6	0.9	12, 11, 12
		2600	5.3	2.3	0.4	8 P, 4 P, 4 P
		5200	8.3	3.1	0.6	11 P, 5 P, 9 P
	MNNG	5.0	2292.3	284.0	171.9	2066, 2200, 2611
TA 100	DMSO	-	32.0	4.0	-	32, 36, 28
	test item	33	30.3	4.5	0.9	35, 30, 26
		100	32.0	3.5	1.0	34, 34, 28
		333	24.7	3.1	0.8	24, 28, 22
		1000	13.3	4.0	0.4	17, 9, 14
		2600	25.3	2.3	0.8	24 P, 28 P, 24 P
		5200	17.3	2.5	0.5	17 P, 15 P, 20 P
	MNNG	5.0	1520.3	241.5	47.5	1371, 1391, 1799
TA 1537	DMSO	-	7.0	1.0	-	8, 6, 7
	test item	33	7.0	2.6	1.0	4, 9, 8
		100	3.7	0.6	0.5	3, 4, 4
		333	6.0	1.0	0.9	7, 5, 6
		1000	5.3	1.5	0.8	4, 5, 7
		2600	3.0	1.0	0.4	3 P, 2 P, 4 P
		5200	4.3	2.5	0.6	7 P, 4 P, 2 P
	AAC	100	1010.0	312.7	144.3	1223, 651, 1156
TA 98	DMSO	-	28.0	1.0	-	27, 29, 28
	test item	33	14.3	4.9	0.5	11, 12, 20
		100	19.7	3.5	0.7	23, 20, 16
		333	19.7	4.7	0.7	16, 18, 25
		1000	9.7	1.5	0.3	8, 10, 11
		2600	11.0	1.0	0.4	12 P, 10 P, 11 P
		5200	9.3	1.5	0.3	8 P, 9 P, 11 P
	NOPD	10	364.7	42.1	13.0	321, 368, 405
E. coli	DMSO	-	59.3	12.4	-	66, 67, 45
	test item	33	54.0	5.0	0.9	54, 49, 59
		100	53.7	9.3	0.9	64, 46, 51
		333	66.3	5.5	1.1	60, 70, 69
		1000	35.3	3.5	0.6	32, 35, 39
		2600	39.3	4.2	0.7	36 P, 38 P, 44 P
		5200	35.7	7.5	0.6	43 P, 36 P, 28 P
	4-NQO	5.0	223.7	34.7	3.8	228, 256, 187

DMSO = dimethyl sulfoxide

MMNG = N-methyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate in DMSO

AAC = 9-aminoacradine 100 µg/plate in DMSO

NOPD = 4-nitro-o-phenylenediamine: 10 µg/plate in DMSO

4-NQO = 4-nitroquinoline-N-oxide 5 μg/plate in DMSO

SD = standard deviation

Table 4: Results of the Prival preincubation test with metabolic activation.

Strain	Test group	Dose (µg/plate)	Mean revertants per plate	SD	Factor	Individual revertant colony counts
TA 1535	DMSO	-	13.7	2.5	-	16, 14, 11
	test item	33	12.0	2.6	0.9	15, 11, 10
		100	10.3	2.1	0.8	8, 12, 11
		333	13.0	1.0	1.0	13, 12, 14
		1000	10.0	3.6	0.7	7, 9, 14
		2600	16.3	3.2	1.2	14 P, 15 P, 20 P
		5200	10.3	3.5	0.8	14 B P, 10 B P, 7 B P
	2-AA	2.5	166.0	47.7	12.1	111, 191, 196
TA 100	DMSO	-	31.7	4.0	-	31, 36, 28
	test item	33	27.7	5.5	0.9	34, 24, 25
		100	31.0	4.0	1.0	31, 35, 27
		333	26.7	3.2	0.8	29, 28, 23
		1000	27.3	5.8	0.9	24, 34, 24
		2600	28.7	3.1	0.9	32 P, 28 P, 26 P
		5200	19.3	1.5	0.6	18 P B, 21 P B, 19 P B
	2-AA	2.5	639.7	82.9	20.2	735, 599, 585
TA 1537	DMSO	-	6.3	1.5	-	6, 5, 8
	test item	33	7.0	3.0	1.1	10, 7, 4
		100	9.0	4.4	1.4	7, 6, 14
		333	8.0	1.7	1.3	7, 10, 7
		1000	8.7	2.1	1.4	8, 11, 7
		2600	1.7	0.6	0.3	2 P, 1 P, 2 P
		5200	2.7	1.5	0.4	1 P, 3 P, 4 P
	2-AA	2.5	134.3	38	21.2	172, 135, 96
TA 98	DMSO	-	25.7	8.7	-	16, 33, 28
	test item	33	24.3	3.1	0.9	21, 27, 25
		100	30.3	4	1.2	31, 26, 34
		333	32.3	7.4	1.3	38, 35, 24
		1000	19.7	3.1	0.8	19, 23, 17
		2600	11.7	2.5	0.5	12 P, 9 P, 14 P
		5200	5.7	0.6	0.2	6 P, 6 P, 5 P
	2-AA	2.5	331.7	18.1	12.9	351, 315, 329
	CoR	210	594.7	201.0	23.2	780, 381, 623
E. coli	DMSO	-	69.0	11.4	-	77, 56, 74
	test item	33	70.0	12.5	1.0	60, 66, 84
		100	73.0	8.5	1.1	72, 82, 65
		333	87.0	12.1	1.3	85, 100, 76
		1000	59.3	6.4	0.9	62, 52, 64
		2600	67.3	1.5	1.0	66 P, 69 P, 67 P
		5200	55.0	5.3	0.8	53 P, 61 P, 51 P
	2-AA	60	557.7	114.5	8.1	685, 525, 463

DMSO = dimethyl sulfoxide

SD = standard deviation

2-AA = 2-aminoanthracene

CoR = Congo Red 210 µg/plate in DMSO

Applicant's summary and conclusion

Conclusions:

The test item was not found to be mutagenic.

Executive summary:

The potential of the test item to induce point mutations by base pair substitutions or frameshifts was evaluated in an experimental study according to the OECD Guideline 471 (1997) and the EU Method B.13 (2998): a reverse mutation (Ames) assay utilising the plate incorporation method performed on Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, as well as Escherichia coli strain WP2 uvrA. The test item (33, 100, 333, 1000, 2600 and 5200 μg/plate) was evaluated for its ability to induce frameshift mutations (S. typhimurium strains TA 98 and TA 1537) and base pair substitution mutations (TA 100 and TA 1535), both with and without the presence of rat liver post-mitochondrial fraction (S9 metabolic activation). Negative (solvent and sterile) and positive controls were tested in parallel; positive controls included

2-aminoanthracene with rat liver S9 mix (2.5 µg/plate in DMSO: TA1535, TA100, TA1537, TA98; and 60 µg/plate in DMSO: E. coli WP2 uvrA), 2-aminoanthracene with hamser liver S9 mix (10 µg/plate in DMSO: TA1535, TA100, TA1537, TA98, E. coli WP2 uvrA), and without S9 mix: 9-aminoacridine (100 µg/plate in DMSO: TA1537), 4-nitro-o-phenylenediamine (10 µg/plate in DMSO: TA98), N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate in DMSO: TA1535, TA100) and Congo Red (210 µg/plate in DMSO: TA 98). As no positive result was found in the first assay, an additional Ames assay was performed using the Pival pre-incubation method, which is specific for azo dyes, using the same test item concentrations and controls.

Precipitation of the test item was found from about 2600 μg/plate onward, both with and without S9 mix. Toxicity in bacteria was occasionally observed depending on the strain and test conditions from about 1000 μg/plate and above. The results of the negative as well as the positive controls fulfilled the acceptance criteria of this study. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the prival preincubation test, either with or without S9 mix.Thus, under the experimental conditions of this study, the test item is considered not mutagenic in theSalmonella typhimurium/Escherichia colireverse mutation assay in the absence and the presence of metabolic activation.