Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2017 - 23 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

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:

Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

EC Number:

916-867-1

Cas Number:

12270-00-7

Molecular formula:

C32H18CoN6O8Na - C32H20CoN8O8Na - C32H22CoN10O8Na

IUPAC Name:

Reaction mass of Sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/137

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.

Method

Species / strain

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:

S9 mix from phenobarbital (i.p.) and β-naphthoflavone (p.o) induced rat liver

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 3125 and 6250 μg/plate (Standard plate test)
Due to the purity of the test substance 6.25 mg/plate was used as top dose.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

With S9 mix, 2.5 μg/plate, dissolved in DMSO (TA 1535, TA 100, TA 1537, TA 98); 60 μg/plate, dissolved in DMSO E.coli WP2uvrA

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix, 5 μg/plate, dissolved in DMSO (TA 1535, TA 100)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine (NOPD)

Remarks:

Without S9 mix, 10 μg/plate, dissolved in DMSO (TA 98)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix, 100 μg/plate, dissolved in DMSO (TA 1537)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix, 5 μg/plate, dissolved in DMSO (E. coli WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in the experiment and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Evaluation criteria:

The test substance 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 substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two expriments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect was observed only with S9 mix at a concentration of 6250 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 1000 μg/plate onward with and without S9 mix.

HISTORICAL CONTROL DATA: Please refer to tables 3 and 4

TOXICITY
A weak bacteriotoxic effect (slight decrease in the number of trp+ revertants) was observed only using the tester strain E. coli with S9 mix at a concentration of 6250 μg/plate.

Any other information on results incl. tables

Table 1: Results without metabolic acitivation ( Standard Plate Test)

Strain	Test group	Dose	Mean	Standard	Factor	Individual revertant colony
		(µg/plate)	revertants per plate	deviation		counts
TA1535	DMSO	-	7.7	0.6	-	7, 8, 8
	Test item	33	9.7	5.5	1.3	7, 6, 16
		100	11.7	5.0	1.5	17, 11, 7
		333	13.7	4.2	1.8	9, 15, 17
		1000	12.0	4.4	1.6	9 P, 17 P, 10 P
		3125	13.7	5.0	1.8	19 P, 13 P, 9 P
		6250	18.7	3.1	2.4	22 P, 16 P, 18 P
	MNNG	5.0	7183.0	372.7	936.9	6753, 7384, 7412
TA100	DMSO	-	116.7	10.3	-	128, 114, 108
	Test item	33	154.7	7.8	1.3	146, 157, 161
		100	226.3	6.0	1.9	227, 232, 220
		333	363.7	22.2	3.1	340, 384, 367
		1000	458.0	53.0	3.9	432 P, 423 P, 519 P
		3125	627.7	22.4	5.4	623 P, 608 P, 652 P
		6250	546.7	59.9	4.7	596 P, 480 P, 564 P
	MNNG	5.0	6016.3	1079.8	51.6	6884, 6358, 4807
TA1537	DMSO	-	13.0	3.5	-	17, 11,11
	Test item	33	26.0	4.4	2.0	29, 28, 21
		100	37.7	5.5	2.9	38, 32, 43
		333	67.0	10.4	5.2	60, 62, 79
		1000	89.7	7.8	6.9	92 P, 81 P, 96 P
		3125	87.7	4.0	6.7	92 P, 87 P, 84 P
		6250	96.3	5.0	7.4	91 P, 97 P, 101 P
	AAC	100	700.7	24.1	53.9	678, 698, 726
TA 98	DMSO	-	26.0	6.9	-	34, 22, 22
	Test item	33	310.0	32.1	11.9	338, 275, 317
		100	587.3	68.3	22.6	575, 661, 526
		333	961.7	59.7	37.0	893, 1001, 991
		1000	871.7	70.1	33.5	823 P, 840 P, 952 P
		3125	919.3	64.0	35.4	948 P, 846 P, 964 P
		6250	1355.3	60.3	52.1	1286 P, 1384 P, 1396 P
	NOPD	10	600.0	27.8	23.1	614, 568, 618
E. COM	DMSO	-	21.7	2.3	-	23, 19, 23
	Test item	33	24.3	0.6	1.1	25, 24, 24
		100	22.7	5.8	1.0	26, 26, 16
		333	26.0	5.0	1.2	21, 31,26
		1000	17.0	1.0	0.8	18 P, 16 P, 17 P
		3125	14.3	2.1	0.7	12 P, 16 P, 15 P
		6250	15.0	1.7	0.7	14 P, 14 P, 17 P
	4-NOO	5	1102.7	93.4	50.9	1081, 1205, 1022

  P=Precipitation

Table 2: Results with metabolic acitivation ( Standard Plate Test)

Strain	Test group	Dose	Mean	Standard	Factor	Individual revertant colony
		(µg/plate)	revertants per plate	deviation		counts
TA 1535	DMSO	-	9.0	2.6	-	8, 7,12
	Test item	33	9.3	4.6	1.0	12, 12, 4
		100	14.7	5.0	1.6	14, 10, 20
		333	12.3	6.7	1.4	18, 14, 5
		1000	10.0	1.7	1.1	9P, 9P, 12P
		3125	14.3	2.5	1.6	17P, 14P, 12P
		6250	10.7	5.7	1.2	17P, 9P, 6P
	2-AA	2.5	245.7	30.7	27.3	280, 221, 236
TA 100	DMSO	-	123.3	13.8	-	118, 113, 139
	Test item	33	183.0	18.5	1.5	176, 169, 204
		100	296.0	19.0	2.4	315, 277, 296
		333	485.3	47.2	3.9	535, 480, 441
		1000	431.7	29.8	3.5	412 P, 466 P,417P
		3125	418.7	57.5	3.4	376 P, 484 P, 396 P
		6250	328.7	6.1	2.7	334 P, 322 P, 330 P
	2-AA	2.5	2669.3	551.1	21.6	2932, 3040, 2036
TA 1537	DMSO	-	7.0	1.0	-	8, 7, 6
	Test item	33	12.0	1.7	1.7	14,11,11
		100	40.7	3.8	5.8	39, 45, 38
		333	84.7	9.6	12.1	76, 95, 83
		1000	53.7	18.9	7.7	39 P, 75 P, 47 P
		3125	58.7	19.6	8.4	77 P, 61 P, 38 P
		6250	62.7	10.7	9.0	65 P, 72 P, 51 P
	2-AA	2.5	170.3	5.5	24.3	165, 170, 176
TA 98	DMSO	_	27.3	6.7	-	35, 23, 24
	Test item	33	251.3	15.1	9.2	258, 234, 262
		100	593.7	88.2	21.7	492, 639, 650
		333	1334.7	33.2	48.8	1330. 1370. 1304
		1000	846.0	9.2	31.0	836 P, 854 P, 848 P
		3125	845.3	12.9	30.9	836 P, 840 P, 860 P
		6250	754.7	8.3	27.6	752 P, 764 P, 748 P
	2-AA	2.5	1878.7	125.5	68.7	1944. 1734. 1958
E. coli	DMSO	-	18.7	1.5	-	20, 17, 19
	Test item	33	21.3	3.5	1.1	18, 21,25
		100	31.0	7.9	1.7	25, 40, 28
		333	26.7	3.1	1.4	24, 26, 30
		1000	16.7	4.7	0.9	22 P, 13 P, 15 P
		3125	14.3	4.5	0.8	14 P, 10 P, 19 P
		6250	8.0	2.6	0.4	9P, 10 P, 5P
	2-AA	60	96.3	4.2	5.2	95, 101. 93

P=Precipitation

Table 3: Historical Positive controls (based on Sorcerer Counter / Ames Study Mananger System (Feb 2016 - Feb 2017)

Strain	S9 Mix	Positive control	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without With	MNNG 2-AA	312 312	110 110	1541 105	6171 520	3967 197	1253.9 56.3
TA 100	Without With	MNNG 2-AA	315 309	110 111	1126 272	5557 3021	3298 1748	1109.6 587.2
TA 1537	Without With	AAC 2-AA	309 312	110 110	253 50	2190 399	1044 141	404.3 50.3
TA98	Without With	NOPD 2-AA	309 315	110 110	324 493	1746 3096	863 1524	206.8 529.1

Table 4: Historical Negative controls (based on Sorcerer Counter / Ames Study Mananger System (Feb 2016 - Feb 2017)

Strain	S9 Mix	Vehicle	No. of Plates	No. of Values	Min	Max	Mean	SD
TA 1535	Without With	All All	414 414	152 152	7 6	16 18	10 10	2.0 2.0
TA 100	Without With	All All	426 417	152 153	71 70	132 147	100 107	11.4 13.7
TA 1537	Without With	All All	411 411	152 152	5 5	13 16	8 9	1.7 2.1
TA98	Without With	All All	420 413	152 152	14 12	34 38	21 28	3.3 4.5
E.coli Without With All All 396 399 152 152 15 17 34 36 24 25 3.9 4.4