[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E.coli WP2 uvra with and without metabolic activation

Gene mutation in mammalian cells (OECD 476, Mouse Lymphoma Assay): negative in L5178Y TK +/- mouse lymphoma cells with and without metabolic activation

Read-across from Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (CAS 2241455 -89 -8).

Chromosome aberration in mammalian cells (OECD 473, Chromosome aberration in vitro): negative in V79 cells with and without metabolic activation

Read-across from Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (CAS 2241455 -89 -8).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May - 05 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains) and trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

First experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation (tested up to the recommended maximum concentration)
Second experiment: 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation (tested up to the recommended maximum concentration)

Vehicle / solvent:

- Vehicle used: deionized water
- Justification for choice of vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria

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 (first experiment); preincubation (second experiment)

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibition, reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed

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 one or more concentrations.
- 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:

Mean numbers of the revertant colonies of each concentration of each test system was calculated and compared to that of the solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

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

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

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

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

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

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant effect on bacterial growth was observed in any strain at any test concentration.

Table 1: Summary Results Experiment I (plate incorporation)

	Test Group		Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
				TA 1535		TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			16 ± 4		10 ± 3	28 ± 2	106 ± 9	50 ± 3
	Untreated			11 ± 4		11 ± 1	33 ± 6	100 ± 8	55 ± 7
	Test item		3 µg	13 ± 4		12 ± 2	29 ± 12	94 ± 13	47 ± 18
			10 µg	16 ± 2		11 ± 3	34 ± 10	104 ± 19	49 ± 10
			33 µg	16 ± 4		10 ± 1	34 ± 8	93 ± 16	52 ± 3
			100 µg	15 ± 5		10 ± 3	30 ± 3	99 ± 8	52 ± 15
			333 µg	12 ± 2		6 ± 2	34 ± 5	92 ± 11	58 ± 10
			1000 µg	11 ± 1		12 ± 4	32 ± 3	98 ± 12	53 ± 10
			2500 µg	10 ± 1		11 ± 2	34 ± 11	93 ± 14	49 ± 9
			5000 µg	16 ± 3		11 ± 1	37 ± 10	88 ± 3	57 ± 17
	NaN3		10 µg	1119 ± 35				1542 ± 75
	4-NOPD		10 µg				405 ± 30
	4-NOPD		50 µg			73 ± 10
	MMS		2.0 µL						963 ± 28
With Activation	Deionised water			11 ± 5		16 ± 4	35 ± 6	114 ± 12	55 ± 10
	Untreated			16 ± 4		16 ± 1	40 ± 12	116 ± 14	62 ± 1
	Test item		3 µg	9 ± 5		15 ± 4	45 ± 6	98 ± 13	51 ± 6
			10 µg	11 ± 3		17 ± 7	36 ± 8	118 ± 16	54 ± 6
			33 µg	11 ± 4		16 ± 4	40 ± 12	115 ± 10	56 ± 11
			100 µg	10 ± 5		16 ± 1	40 ± 6	110 ± 6	56 ± 16
			333 µg	13 ± 4		15 ± 2	43 ± 9	94 ± 12	58 ± 8
			1000 µg	9 ± 3		20 ± 6	38 ± 3	96 ± 14	59 ± 7
			2500 µg	10 ± 1		14 ± 6	41 ± 15	105 ± 26	61 ± 4
			5000 µg	10 ± 4		18 ± 4	40 ± 7	85 ± 5	53 ± 5
	2-AA		2.5 µg	325 ± 24		417 ± 10	3168 ± 188	2727 ± 207
	2-AA		10.0 µg						306 ± 12
NaN3 2-AA 4-NOPD MMS		sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Table 2: Summary Results Experiment II (pre incubation)

Metabolic Activation	Test Group		Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
				TA 1535		TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionionised water			17 ± 6		15 ± 6	30 ± 4	102 ± 16	52 ± 9
	Untreated			15 ± 3		18 ± 1	36 ± 3	113 ± 16	55 ± 4
	Test item		33 µg	13 ± 3		14 ± 4	34 ± 9	122 ± 14	60 ± 7
			100 µg	15 ± 4		16 ± 7	41 ± 2	108 ± 11	56 ± 10
			333 µg	15 ± 0		15 ± 6	32 ± 8	106 ± 17	64 ± 5
			1000 µg	14 ± 6		16 ± 3	31 ± 10	113 ± 9	61 ± 4
			2500 µg	13 ± 4		17 ± 3	33 ± 2	116 ± 5	58 ± 9
			5000 µg	16 ± 6		13 ± 3	33 ± 9	103 ± 11	56 ± 7
	NaN3		10 µg	1290 ± 5				1603 ± 111
	4-NOPD		10 µg				447 ± 10
	4-NOPD		50 µg			86 ± 4
	MMS		2.0 µL						760 ± 31
With Activation	Deionionised water			16 ± 4		17 ± 6	44 ± 8	110 ± 9	61 ± 4
	Untreated			10 ± 2		15 ± 2	46 ± 6	112 ± 3	63 ± 9
	Test item		33 µg	13 ± 1		16 ± 4	41 ± 14	108 ± 2	64 ± 17
			100 µg	15 ± 7		16 ± 5	47 ± 6	103 ± 6	68 ± 9
			333 µg	15 ± 6		15 ± 4	44 ± 5	110 ± 9	60 ± 3
			1000 µg	12 ± 3		19 ± 4	48 ± 4	102 ± 1	61 ± 1
			2500 µg	14 ± 2		16 ± 6	45 ± 3	95 ± 11	54 ± 6
			5000 µg	13 ± 4		16 ± 4	47 ± 8	110 ± 3	59 ± 7
	2-AA		2.5 µg	308 ± 13		265 ± 6	2865 ± 116	3148 ± 186
	2-AA		10.0 µg						366 ± 1
NaN3 2-AA 4-NOPD MMS		sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Table 3: Individual Results of Experiment I (Without metabolic activation)

Strain	Compound		Dose level per plate	Mean revertants per plate		Standard Deviation	Ratio treated / solvent	Individual revertant colony counts
TA 1535	Test item		3 µg	13.3		3.8	0.8	15, 16, 9
			10 µg	15.7		1.5	1.0	16, 14, 17
			33 µg	15.7		4.2	1.0	19, 17, 11
			100 µg	14.7		5.1	0.9	9, 19, 16
			333 µg	12.0		2.0	0.7	12, 14, 10
			1000 µg	10.7		0.6	0.7	11, 11, 10
			2500 µg	10.3		0.6	0.6	10, 11, 10
			5000 µg	16.3		3.2	1.0	15, 20, 14
	Deionised water			16.3		4.0		14, 21, 14
	Untreated Control			11.0		4.0		7, 15, 11
TA 1537	Test item		3 µg	12.3		1.5	1.3	14, 12, 11
			10 µg	11.0		2.6	1.1	10, 9, 14
			33 µg	9.7		1.2	1.0	9, 11, 9
			100 µg	9.7		2.5	1.0	12, 7, 10
			333 µg	6.3		2.3	0.7	5, 9, 5
			1000 µg	12.0		4.4	1.2	7, 14, 15
			2500 µg	11.3		2.3	1.2	10, 10, 14
			5000 µg	10.7		0.6	1.1	11, 11, 10
	Deionised water			9.7		2.5		7, 12, 10
	Untreated Control			10.7		1.2		10, 12, 10
TA 98	Test item		3 µg	29.0		12.1	1.0	22, 22, 43
			10 µg	34.0		10.1	1.2	32, 25, 45
			33 µg	34.3		8.3	1.2	37, 25, 41
			100 µg	30.3		3.1	1.1	31, 27, 33
			333 µg	34.3		5.1	1.2	30, 33, 40
			1000 µg	31.7		2.9	1.1	30, 35, 30
			2500 µg	34.3		10.7	1.2	32, 46, 25
			5000 µg	37.3		10.0	1.3	26, 41, 45
	Deionised water			28.0		2.0		26, 30, 28
	Untreated Control			33.0		6.1		36, 26, 37
TA 100	Test item		3 µg	93.7		12.9	0.9	99, 79, 103
			10 µg	104.0		19.2	1.0	113, 117, 82
			33 µg	92.7		15.6	0.9	91, 109, 78
			100 µg	99.3		7.6	0.9	96, 108, 94
			333 µg	91.7		10.6	0.9	82, 103, 90
			1000 µg	98.0		12.2	0.9	106, 84, 104
			2500 µg	92.7		14.2	0.9	108, 80, 90
			5000 µg	87.7		2.5	0.8	88, 85, 90
	Deionised water			105.7		9.0		101, 116, 100
	Untreated Control			100.3		7.8		94, 98, 109
WP2	Test item		3 µg	47.0		18.2	0.9	68, 37, 36
uvrA			10 µg	49.3		10.0	1.0	38, 53, 57
			33 µg	52.0		2.6	1.0	49, 53, 54
			100 µg	52.0		15.1	1.0	69, 47, 40
			333 µg	57.7		10.1	1.2	47, 59, 67
			1000 µg	53.3		10.0	1.1	57, 42, 61
			2500 µg	49.0		8.5	1.0	41, 58, 48
			5000 µg	56.7		16.8	1.1	53, 75, 42
	Deionised water			50.0		2.6		47, 52, 51
	Untreated Control			54.7		7.0		48, 54, 62
TA 1535	NaN3		10 µg	1119.3		35.2	68.5	1152, 1124, 1082
TA 1537	4-NOPD		50 µg	73.0		10.1	7.6	82, 62, 75
TA 98	4-NOPD		10 µg	405.3		29.8	14.5	425, 420, 371
TA 100	NaN3		10 µg	1542.0		75.4	14.6	1629, 1500, 1497
WP2 uvrA	MMS		2.0 µL	962.7		28.2	19.3	985, 972, 931
NaN3 4-NOPD MMS		sodium azide 4-nitro-o-phenylene-diamine methyl methane sulfonate

 

Table 4: Individual Results of Experiment I (With metabolic activation)

Strain		Compound			Dose level per plate	Mean revertants per plate		Standard Deviation	Ratio treated / solvent	Individual revertant colony counts
TA 1535		Test item			3 µg	9.3		4.9	0.8	15, 6, 7
					10 µg	11.3		2.5	1.0	11, 14, 9
					33 µg	11.0		3.6	1.0	7, 14, 12
					100 µg	9.7		4.6	0.9	7, 15, 7
					333 µg	13.0		3.6	1.1	17, 10, 12
					1000 µg	9.0		3.0	0.8	12, 6, 9
					2500 µg	9.7		0.6	0.9	9, 10, 10
					5000 µg	10.0		4.0	0.9	10, 14, 6
		Deionised water				11.3		5.1		17, 10, 7
		Untreated Control				15.7		4.2		17, 11, 19
TA 1537		Test item			3 µg	15.3		4.0	1.0	19, 16, 11
					10 µg	17.0		7.0	1.1	9, 22, 20
					33 µg	16.3		4.0	1.0	14, 21, 14
					100 µg	16.0		1.0	1.0	17, 16, 15
					333 µg	15.3		1.5	1.0	15, 14, 17
					1000 µg	19.7		5.5	1.3	14, 25, 20
					2500 µg	13.7		6.4	0.9	10, 10, 21
					5000 µg	18.0		3.6	1.1	14, 19, 21
		Deionised water				15.7		4.0		12, 20, 15
		Untreated Control				15.7		1.2		17, 15, 15
TA 98		Test item			3 µg	45.0		6.1	1.3	41, 42, 52
					10 µg	36.3		7.6	1.0	43, 38, 28
					33 µg	39.7		12.4	1.1	32, 33, 54
					100 µg	40.3		6.4	1.1	43, 45, 33
					333 µg	42.7		9.3	1.2	47, 49, 32
					1000 µg	37.7		2.5	1.1	38, 35, 40
					2500 µg	41.0		14.7	1.2	58, 32, 33
					5000 µg	40.0		7.0	1.1	47, 40, 33
		Deionised water				35.3		5.9		31, 33, 42
		Untreated Control				40.0		11.5		51, 28, 41
TA 100		Test item			3 µg	97.7		13.1	0.9	84, 99, 110
					10 µg	117.7		15.6	1.0	134, 103, 116
					33 µg	115.0		9.5	1.0	125, 106, 114
					100 µg	109.7		5.5	1.0	110, 115, 104
					333 µg	94.3		12.1	0.8	90, 85, 108
					1000 µg	96.0		14.4	0.8	100, 108, 80
					2500 µg	105.0		26.0	0.9	75, 121, 119
					5000 µg	85.3		4.9	0.7	82, 91, 83
		Deionised water				114.3		11.6		122, 120, 101
		Untreated Control				116.0		13.7		113, 104, 131
WP2		Test item			3 µg	50.7		5.5	0.9	56, 51, 45
uvrA					10 µg	53.7		5.9	1.0	56, 47, 58
					33 µg	56.0		11.4	1.0	64, 61, 43
					100 µg	55.7		15.9	1.0	74, 46, 47
					333 µg	58.0		7.5	1.1	51, 66, 57
					1000 µg	58.7		6.7	1.1	53, 66, 57
					2500 µg	60.7		3.5	1.1	64, 57, 61
					5000 µg	53.0		5.0	1.0	48, 58, 53
		Deionised water				54.7		9.8		49, 66, 49
		Untreated Control				62.3		0.6		63, 62, 62
TA 1535	2-AA			2.5 µg		325.0		23.6	28.7	345, 299, 331
TA 1537	2-AA			2.5 µg		416.7		9.7	26.6	406, 419, 425
TA 98	2-AA			2.5 µg		3168.3		188.3	89.7	3143, 2994, 3368
TA 100	2-AA			2.5 µg		2726.7		207.1	23.8	2910, 2768, 2502
WP2 uvrA	2-AA			10.0 µg		306.3		11.6	5.6	312, 293, 314
2-AA			2-aminoanthracene

 

Table 5: Individual Results of Experiment II (Without metabolic activation)

Strain	Compound		Dose level per plate	Mean revertants per plate		Standard Deviation	Ratio treated / solvent	Individual revertant colony counts
TA 1535	Test item		33 µg	12.7		3.2	0.7	14, 9, 15
			100 µg	15.0		4.0	0.9	11, 15, 19
			333 µg	15.0		0.0	0.9	15, 15, 15
			1000 µg	13.7		5.5	0.8	10, 20, 11
			2500 µg	13.0		3.6	0.8	12, 17, 10
			5000 µg	15.7		6.0	0.9	15, 22, 10
	Deionised water			17.0		6.2		22, 19, 10
	Untreated Control			14.7		2.5		12, 15, 17
TA 1537	Test item		33 µg	14.0		3.6	1.0	10, 15, 17
			100 µg	16.0		6.6	1.1	9, 22, 17
			333 µg	14.7		5.5	1.0	9, 15, 20
			1000 µg	16.3		2.5	1.1	19, 16, 14
			2500 µg	17.3		2.9	1.2	14, 19, 19
			5000 µg	12.7		3.2	0.9	9, 15, 14
	Deionised water			14.7		6.0		14, 9, 21
	Untreated Control			17.7		1.2		17, 19, 17
TA 98	Test item		33 µg	34.0		8.5	1.1	25, 42, 35
			100 µg	41.3		1.5	1.4	40, 43, 41
			333 µg	31.7		8.1	1.0	41, 26, 28
			1000 µg	31.3		9.8	1.0	20, 37, 37
			2500 µg	33.3		1.5	1.1	33, 32, 35
			5000 µg	33.3		9.3	1.1	36, 41, 23
	Deionised water			30.3		4.0		28, 35, 28
	Untreated Control			35.7		2.5		36, 33, 38
TA 100	Test item		33 µg	122.0		13.9	1.2	115, 138, 113
			100 µg	107.7		11.0	1.1	114, 114, 95
			333 µg	105.7		16.7	1.0	119, 87, 111
			1000 µg	113.3		8.6	1.1	104, 121, 115
			2500 µg	116.3		5.0	1.1	121, 111, 117
			5000 µg	103.3		11.2	1.0	113, 106, 91
	Deionised water			102.0		16.1		85, 104, 117
	Untreated Control			113.3		16.2		132, 104, 104
WP2	Test item		33 µg	60.3		7.0	1.2	53, 67, 61
uvrA			100 µg	56.0		10.0	1.1	66, 46, 56
			333 µg	64.0		5.0	1.2	59, 64, 69
			1000 µg	60.7		4.2	1.2	64, 56, 62
			2500 µg	57.7		9.3	1.1	62, 64, 47
			5000 µg	55.7		6.5	1.1	62, 56, 49
	Deionised water			51.7		9.1		45, 48, 62
	Untreated Control			54.7		4.0		59, 54, 51
TA 1535	NaN3		10 µg	1290.0		5.2	75.9	1287, 1296, 1287
TA 1537	4-NOPD		50 µg	86.0		3.6	5.9	87, 89, 82
TA 98	4-NOPD		10 µg	447.3		10.2	14.7	443, 440, 459
TA 100	NaN3		10 µg	1603.0		110.7	15.7	1710, 1489, 1610
WP2 uvrA	MMS		2.0 µL	760.3		30.7	14.7	785, 726, 770
NaN3 4-NOPD MMS		sodium azide 4-nitro-o-phenylene-diamine methyl methane sulfonate

 

Table 6: Individual Results of Experiment II (With metabolic activation)

Strain	Compound			Dose level per plate	Mean revertants per plate		Standard Deviation	Ratio treated / solvent	Individual revertant colony counts
TA 1535	Test item			33 µg	12.7		1.2	0.8	12, 14, 12
				100 µg	15.3		6.5	1.0	9, 15, 22
				333 µg	15.3		5.5	1.0	10, 15, 21
				1000 µg	11.7		3.1	0.7	9, 15, 11
				2500 µg	14.0		2.0	0.9	16, 12, 14
				5000 µg	13.0		3.6	0.8	17, 10, 12
	Deionised water				16.0		3.6		19, 12, 17
	Untreated Control				10.3		1.5		9, 12, 10
TA 1537	Test item			33 µg	15.7		4.0	0.9	20, 12, 15
				100 µg	16.3		4.5	1.0	16, 21, 12
				333 µg	14.7		4.0	0.9	11, 19, 14
				1000 µg	19.0		3.6	1.1	20, 15, 22
				2500 µg	15.7		6.0	0.9	22, 10, 15
				5000 µg	15.7		4.2	0.9	11, 19, 17
	Deionised water				17.0		6.0		17, 23, 11
	Untreated Control				15.3		1.5		15, 17, 14
TA 98	Test item			33 µg	41.3		13.6	0.9	43, 27, 54
				100 µg	47.3		6.0	1.1	48, 53, 41
				333 µg	43.7		4.9	1.0	47, 38, 46
				1000 µg	48.3		4.2	1.1	45, 47, 53
				2500 µg	45.3		2.5	1.0	48, 45, 43
				5000 µg	46.7		7.8	1.1	38, 53, 49
	Deionised water				43.7		8.1		51, 45, 35
	Untreated Control				46.0		6.1		42, 53, 43
TA 100	Test item			33 µg	108.3		2.1	1.0	110, 106, 109
				100 µg	103.3		5.8	0.9	100, 100, 110
				333 µg	109.7		9.1	1.0	120, 103, 106
				1000 µg	101.7		1.2	0.9	103, 101, 101
				2500 µg	95.3		11.0	0.9	104, 99, 83
				5000 µg	110.0		2.6	1.0	113, 108, 109
	Deionised water				110.0		8.9		113, 100, 117
	Untreated Control				112.0		2.6		110, 111, 115
WP2	Test item			33 µg	64.3		16.6	1.1	66, 47, 80
uvrA				100 µg	68.0		8.7	1.1	78, 63, 63
				333 µg	59.7		3.2	1.0	62, 61, 56
				1000 µg	61.3		0.6	1.0	62, 61, 61
				2500 µg	54.3		6.1	0.9	49, 53, 61
				5000 µg	58.7		6.7	1.0	53, 66, 57
	Deionised water				60.7		3.5		61, 64, 57
	Untreated Control				63.3		8.7		61, 73, 56
TA 1535	2-AA		2.5 µg		307.7		13.4	19.2	323, 302, 298
TA 1537	2-AA		2.5 µg		264.7		5.8	15.6	268, 268, 258
TA 98	2-AA		2.5 µg		2864.7		116.5	65.6	2994, 2768, 2832
TA 100	2-AA		2.5 µg		3147.7		186.5	28.6	3039, 3363, 3041
WP2 uvrA	2-AA		10.0 µg		366.3		1.2	6.0	365, 367, 367
2-AA		2-aminoanthracene

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five tester strains (TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Refer to analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

source CAS 2241455-89-8

Conclusions:

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their in vitro genetic toxicity potential. Based on the results of the available chromosome aberration assay, the source substance (CAS 2241455-89-8) was not considered to induce chromosomal abrrations in Chinese hamster lung fibroblasts (V79). Applying the read-across approach, the target substance reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate (EC 950-225-1) is expected to be non-clastogenic in the chromosome aberration assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

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:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

source CAS 2241455-89-8

Conclusions:

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their in vitro genetic toxicity potential. Based on the results of the available mouse lymphoma assay, the source substance, CAS 2241455-89-8 was not considered to be mutagenic. Applying the read-across approach, the target substance reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate (EC 950-225-1) is not expected to be mutagenic in mouse lymphoma cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Mutagenicity in bacteria

The in-vitro genetic toxicity of the Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate (EC 950-225-1) was assessed in a bacterial reverse mutation assay (Ames test) according to OECD TGuideline 471 and GLP criteria ( 2019). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains TA 98, 100, 1535, 1537 and E.coli WP2uvra at concentrations up to 5000 µg/plate in 2 independent experiments (plate incorporation and pre-incubation method). The test substance did not induce an increase in reversions in any of the tested strains with or without metabolic activation. Cytotoxicity was not observed. The vehicle and positive controls proved the validity of the experiment. Thus the test substance Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate was not considered mutagenic in bacteria.

 

Justification for read-across

There are no experimental data available regarding the mutagenicity and cytogenicity in mammalian cells for Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate (EC 950-225-1). Thus, read-across from an appropriate analogue substance Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid (CAS 2241455-89-8) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfill the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4. . The read-across is based on common (bio)transformation compounds of source and target substance.A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Mutagenicity in mammalian cells

The potential mutagenicity of the analogue substance Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed in a GLP-compliant study conducted according to OECD Guideline 476 (2013). No cytotoxicity was observed with or without S9-mix after treatment with up to 10 mM test substance for 4 h in a range-finding assay. In the main experiment L5178Y TK +/- mouse lymphoma cells were treated with the test material with and without metabolic activation system (S9 mix) in two independent experiments. Vehicle (RPMI medium + horse serum) and positive controls were included. In the first experiment, cells were exposed to the test material for 4 h in the presence and absence of S9 mix at concentrations of 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, and 10 mM. In the second experiment, cells were treated for 4 h with metabolic activation and for 24 h without metabolic activation at concentrations of 0.8, 2, 4, 6, 7, 8, 9, and 10 mM.

No cytotoxic effects were noted in the presence and absence of S9-mix. The test material did not induce any toxicologically significant concentration-related increases in the mutant frequency at any concentration, either with or without metabolic activation. No precipitation was observed up to the maximum concentration of 10 mM. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system.

The test material was thus considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

The target and source substances are considered unlikely to differ in their mutagenic potential in mammalian cells. Therefore, Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate (EC 950-225-1) was considered non-mutagenic to L5178Y cells.

 

Clastogenicity in vitro

Clastogenic effects of the analogue substance Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid were assessed in a chromosome aberration test in V79 cells conducted according to OECD Guideline 473 (2013). Chinese hamster fibroblasts (V79) were exposed to 0.16 - 10 mM or 0.31 - 10 mM test substance in the presence and in the absence of metabolic activation, respectively. For microscopic analysis 2.5, 5, and 10 mM (Experiment I and II without S9; Experiment I with S9) and 3, 6, and 10 mM (Experiment 2 with S9) were chosen.Positive and negative (medium) control cultures were included in each assay and historical control data were provided. No precipitation and no cytotoxicity was observed at any concentration.

In experiment I without metabolic activation the aberration rate of the negative control (1.5%) and all dose groups treated with the test item (2.5 mM and 10 mM 1% and 5 mM 2%) were within the historical control data of the testing facility (0.0% – 4.0%).

With metabolic activation, the aberration rates of the negative control (3.5%) and the treated dose groups 4 and 6 at 2.5 mM and 10 mM (2.0%) were within the historical controls. An increase of aberrant cells was noted at a concentration of 5.0 mM (5% chromosome aberration). Based on the increased single value in experiment I a clear assessment was not possible. To verify the observed effects an independent repetition of the experiment was performed (second experiment with and without metabolic activation).

In experiment II without metabolic activation the aberration rate of the negative control (1.5%) and all dose groups treated with the test item (2.5 mM (1.0%), 5.0 mM (0.0%) and 10 mM (2.5%)) were within the historical control data of the testing facility (0.0% – 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. Herewith the increase of the aberration rate in experiment I was not confirmed in experiment II.

With metabolic activation the aberration rates of the negative control (2.5%) and all dose groups treated with the test item (3.0 mM and 6.0 mM (1.5%), and 10.0 mM (2.5%)) were within the historical control data of the testing facility (0.0% – 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed. Herewith the increase of the aberration rate in experiment I was not confirmed in experiment II.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

In conclusion, the test item is considered to be non-clastogenic under the conditions of the experiment. 

 

The target and source substances are considered unlikely to differ in their clastogenic potential in mammalian cells. Therefore, Reaction mass of dipotassium 2-(3,4-dimethyl-1H-pyrazol-1-yl) succinate and dipotassium 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinate (EC 950-225-1) was considered to be non-clastogenic.

Justification for classification or non-classification

The available and read-across data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.