Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

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:

2018-08-23 till 2018-09-03

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:

Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

EC Number:

233-357-8

EC Name:

Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

Cas Number:

10127-27-2

Molecular formula:

C16H11CrN5O8S.Na

IUPAC Name:

sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

Method

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:

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
Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

Vehicle / solvent:

Solvent used: deionized water
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

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.

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:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

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

Test results 5

Key result

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:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: soluble (solvent)
Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. No precipitation of the test item occurred in the overlay agar on the incubated agar plates
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Any other information on results incl. tables

Summary of Experiment I

Study Name: 1916800	Study Code: Envigo 1916800
Experiment: 1916800 VV Plate	Date Plated: 23.08.2018
Assay Conditions:	Date Counted: 27.08.2018

Metabolic Activation	Test Group	Dose Level (per plate)		TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537Revertant Colony Counts (Mean ±SD)	TA 98Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without Activation	Deionized water			8 ± 2	13 ± 3	25 ± 6	98 ± 2	36 ± 8
	Untreated			17 ± 6	10 ± 2	23 ± 8	103 ± 18	36 ± 4
	Aluminium	3 µg		8 ± 3	15 ± 1	22 ± 7	115 ± 18	33 ± 10
	Orange G	10 µg		9 ± 3	12 ± 4	24 ± 7	107 ± 9	36 ± 11
		33 µg		11 ± 1	9 ± 2	25 ± 7	120 ± 25	32 ± 8
		100 µg		12 ± 2	12 ± 3	21 ± 6	97 ± 13	31 ± 1
		333 µg		9 ± 3	13 ± 3	23 ± 3	106 ± 25	36 ± 9
		1000 µg		12 ± 2	11 ± 4	24 ± 5	98 ± 6	36 ± 3
		2500 µg		9 ± 3	11 ± 3	29 ± 3	72 ± 5	30 ± 9
		5000 µg		12 ± 3	9 ± 1	32 ± 8	62 ± 21	30 ± 8
	NaN3	10 µg		979 ± 39			1271 ± 78
	4-NOPD	10 µg				536 ± 16
	4-NOPD	50 µg			90 ± 8
	MMS	2.0 µL						855 ± 29
With Activation	Deionized water			12 ± 4	18 ± 2	43 ± 8	109 ± 14	39 ± 3
	Untreated			12 ± 2	19 ± 5	33 ± 4	129 ± 7	46 ± 5
	Aluminium	3 µg		13 ± 5	15 ± 7	33 ± 0	118 ± 5	41 ± 5
	Orange G	10 µg		9 ± 2	12 ± 2	36 ± 6	115 ± 4	37 ± 12
		33 µg		12 ± 2	12 ± 4	32 ± 2	127 ± 18	41 ± 5
		100 µg		13 ± 2	12 ± 2	41 ± 13	131 ± 14	41 ± 12
		333 µg		10 ± 2	11 ± 5	39 ± 6	126 ± 22	54 ± 12
		1000 µg		12 ± 4	14 ± 6	30 ± 1	115 ± 5	41 ± 6
		2500 µg		14 ± 2	11 ± 3	34 ± 8	100 ± 19	41 ± 4
		5000 µg		12 ± 5	12 ± 2	25 ± 7	69 ± 15	22 ± 4
	2-AA	2.5 µg		217 ± 29	324 ± 26	2327 ± 280	2925 ± 194
	2-AA	10.0 µg						208 ± 29

Summary of Experiment II

Study Name: 1916800	Study Code: Envigo 1916800
Experiment: 1916800 HV2 Pre	Date Plated: 30.08.2018
Assay Conditions:	Date Counted: 03.09.2018

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 Activation	Deionized water			13 ± 3	9 ± 1	25 ± 3	98 ± 9	43 ± 2
	Untreated			10 ± 4	12 ± 3	30 ± 11	105 ± 21	43 ± 3
	Aluminium	33 µg		12 ± 5	10 ± 5	30 ± 7	102 ± 3	38 ± 7
	Orange G	100 µg		14 ± 3	11 ± 1	24 ± 2	100 ± 10	45 ± 2
		333 µg		11 ± 3	11 ± 5	28 ± 3	93 ± 8	39 ± 2
		1000 µg		10 ± 3	12 ± 2	26 ± 5	106 ± 8	45 ± 7
		2500 µg		10 ± 5	13 ± 4	23 ± 6	99 ± 21	49 ± 2
		5000 µg		7 ± 2	12 ± 4	29 ± 8	44 ± 3	47 ± 5
	NaN3	10 µg		1007 ± 34			1801 ± 133
	4-NOPD	10 µg				986 ± 70
	4-NOPD	50 µg			80 ± 5
	MMS	2 µL						838 ± 31
With Activation	Deionized water			11 ± 2	20 ± 7	45 ± 7	143 ± 7	47 ± 9
	Untreated			16 ± 4	29 ± 4	53 ± 9	127 ± 11	50 ± 4
	Aluminium	33 µg		15 ± 5	22 ± 10	55 ± 7	143 ± 2	52 ± 12
	Orange G	100 µg		14 ± 4	22 ± 3	48 ± 9	143 ± 4	54 ± 5
		333 µg		14 ± 3	21 ± 9	48 ± 7	137 ± 16	40 ± 3
		1000 µg		15 ± 4	19 ± 7	52 ± 11	136 ± 8	42 ± 6
		2500 µg		16 ± 1	18 ± 5	37 ± 8	70 ± 6	35 ± 3
		5000 µg		11 ± 3	11 ± 3	37 ± 12	65 ± 19	35 ± 10
	2-AA	2.5 µg					2982 ± 36
	2-AA	2.5 µg		361 ± 18	302 ± 22
	2-AA	10 µg						1193 ± 17
	Congo red	500 µg				439 ± 150

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Congo Red

Applicant's summary and conclusion

Conclusions:

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 genomes 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:

The test item 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.

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 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 II:                           33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate. No precipitation of the test itemoccurredin the overlay agar on the incubated agar plates

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 the test groups with and without metabolic activation.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration 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. They showed a distinct increase in induced revertant colonies.