Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

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:

13 Nov 2017 - 9 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 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)

Remarks:

GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit, Schwabach, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

EC Number:

257-479-6

EC Name:

Dipotassium dihydrogen 4-[5-[3-carboxylato-5-hydroxy-1-(4-sulphonatophenyl)-1H-pyrazol-4-yl]penta-2,4-dienylidene]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

Cas Number:

51858-17-4

Molecular formula:

C25H16N4O12S2.2K

IUPAC Name:

dipotassium dihydrogen 4-{5-[3-carboxylato-5-oxo-1-(4-sulfonatophenyl)-1,5-dihydro-4H-pyrazol-4-ylidene]penta-1,3-dien-1-yl}-5-hydroxy-1-(4-sulfonatophenyl)-1H-pyrazole-3-carboxylate

Test material form:

solid: crystalline

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 98 and TA 100

Experiment: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The test substance concentrations used in the main experiment were chosen according to the results of the pre-experiment.

Vehicle / solvent:

- Vehicle/solvent used: Distilled water
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 acitivity.

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 (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (pre-experiment and main experiment 1) and pre-incubation test (experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants down to a mutation factor of approximately ≤ 0.5 or a clearing of the bacterial background lawn

POSITIVE CONTROL 2-AA:
- Proficiency of the activity of S9 was determined and confirmed prior to the experiment

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant count by the mean values of the solvent control (the exact and not rounded values are used for calculation)
A test item is considered mutagenic if:
- a clear dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabloic activation.

A biologically relevant increase is describes as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: Only strain TA 98 and TA 100 were tested in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects of the test item was observed in any strain.

Any other information on results incl. tables

Table 1: Summary of Pre-Experiment

Substance	Dose (µg/plate)	TA98 Mutation Factor (toxicity*)		TA 100 Mutation Factor (toxicity*)
		without S9	with S9	without S9	with S9
Solvent Control (Distilled water)		1.0	1.0	1.0	1.0
4-NOPD	10.0	12.4	-	-	-
NaN3	10.0	-	-	6.7	-
2-AA	2.50	-	58.7	-	13.8
Test Item	3.16	0.8	0.9	0.9	0.9
	10.0	0.9	0.7	0.9	1.0
	31.6	0.8	0.9	0.9	0.9
	100	0.8	0.8	0.9	0.9
	316	0.8	0.6	0.8	0.8
	1000	0.8	0.8	0.8	1.0
	2500	0.9	1.0	1.0	1.0
	5000	0.9	0.7	1.1	1.1

* toxicity parameter: B = Background lawn reduced, N = No background lawn

 

Table 2: Experiment 1 (Plate-incorporation Test)

	EXPERIMENT 1 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Distilled water		42 ± 0.6	94 ± 7.0	22±10.3	22±2.1	284±17.6
Test Item	31.6	35 ± 1.7	86 ± 10.0	24±3.6	31±5.0	275±28.0
	100	33 ± 7.1	84 ± 1.2	28±2.5	22±7.0	261±14.0
	316	35 ± 7.1	79 ± 6.4	23±2.6	28±2.6	268±35.7
	1000	35 ± 6.0	74 ± 4.6	21±6.7	20±1.2	280±23.9
	2500	39 ± 9.2	93 ± 3.5	16±4.2	23±3.1	265±13.2
	5000	37 ± 7.2	101 ± 12.7	18±4.0	24±2.5	257±55.4
4-NOPD	10 (TA 98)/40 (TA 1537)	524 ± 32.9	-	-	234±10.3	-
NaN3	10	-	631 ± 41.1	1155±2.9	-	-
MMS	1					1915±320.4
	S9-Mix	With
	Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Distilled water	-	34 ± 4.4	89±9.5	14±1.5	27±4.0	318±25.4
	31.6	31 ± 6.7	80±4.6	19±1.2	31±6.1	320±12.7
	100	26 ± 7.8	76±5.3	21±1.0	30±4.6	330±45.6
	316	22 ± 2.0	71±1.5	17±3.6	26±8.7	320±13.1
	1000	26 ± 5.9	87±25.4	14±7.0	23±2.5	368±34.1
	2500	35 ± 4.4	85±21.2	18±12.5	21±1.0	373±18.0
	5000	25 ± 2.3	95±2.0	13±4.7	24±3.2	348±27.6
2-AA	2.5/ 10 (only TA 102)	1994 ± 258.6	1225±45.7	268±10.1	225±58.0	955±151.7
	NC = Negative/Vehicle Control PC = Respective positive control substances (for details see method description) SD = Standard Deviation

Table 3: Experiment 2 (Pre-incubation Test)

	EXPERIMENT 2 (Revertant colonies per plate ± SD)
	S9-Mix	Without
	Test item (µg/plate)	TA 98	TA 100		TA 1535	TA 1537	TA 102
Distilled water		20 ± 3.1	128 ± 6.4		18 ± 3.0	21 ± 3.1	250 ± 35.4
Test Item	31.6	19 ± 4.0	105 ± 6.1		19 ± 2.0	21 ± 2.1	267 ± 38.8
	100	18 ± 3.5	114 ± 9.1		16 ± 1.5	20 ± 3.0	207 ± 25.5
	316	19 ± 6.6	115 ± 9.0		19 ± 1.5	20 ± 1.0	184 ± 16.8
	1000	19 ± 4.6	115 ± 18.2		20 ± 2.5	24 ± 0.6	213 ± 1.0
	2500	18 ± 2.1	114 ± 27.1		17 ± 2.1	22 ± 1.5	234 ± 5.9
	5000	20 ± 3.1	119 ± 23.4		20 ±.5	21 ± 2.6	219 ± 29.2
4-NOPD	10 (TA 98)/40 (TA 1537)	342 ± 27.6	-		-	115 ± 15.6	-
NaN3	10	-	590 ± 66.5		268 ± 116.3	-	-
MMS	1	-	-		-	-	1296 ± 213.2
	S9-Mix	With
	Test item (µg/plate)	TA 98		TA 100	TA 1535	TA 1537	TA 102
Distilled water	-	18 ± 4.5		117 ± 23.1	14 ± 3.6	17 ± 2.6	220 ± 40.8
	31.6	20 ± 3.5		100 ± 12.7	15 ± 2.5	18 ± 3.1	239 ± 1.7
	100	18 ± 2.1		99 ± 21.0	13 ± 1.5	18 ± 1.5	240 ± 4.6
	316	19 ± 6.4		100 ± 16.3	15 ± 0.6	20 ± 1.0	239 ± 0.0
	1000	17 ± 1.7		101 ± 20.3	15 ± 1.5	19 ± 2.5	241 ± 3.1
	2500	21 ± 2.3		104 ± 21.0	14 ± 2.0	21 ± 3.8	255 ± 5.1
	5000	18 ± 2.5		109 ± 13.4	15 ± 2.0	19 ± 2.1	262 ± 1.2
2-AA	2.5/ 10 (only TA 102)	215 ± 9.7		420 ± 0.6	122 ± 21.9	151 ± 20.3	838 ± 23.8
	NC = Negative/Vehicle Control PC = Respective positive control substances (for details see method description) SD = Standard Deviation

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the item was not mutagenic in any of the five strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102) tested with and without metabolic activation up to 5000 µg/plate.