sodium 3-((2,3-dicyanophenyl)sulfonyl)propane-1-sulfonate

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:

The experimental phase started on 21 November 2013 and ended on 19 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was carried out, however, as the test item was formulated within four hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium 3-((2,3-dicyanophenyl)sulfonyl)propane-1-sulfonate

EC Number:

696-126-4

Cas Number:

886592-17-2

Molecular formula:

C11H9N2O5S2Na

IUPAC Name:

sodium 3-((2,3-dicyanophenyl)sulfonyl)propane-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

Histidine for Salmonella
Tryptophan for E. Coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not Applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/betanaphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Experiment one: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Concentrations relate to active as formulated concentrations were adjusted to allow for the water and impurity content (5.12 % w/w) of the test material.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 ug/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2-ug/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2ug/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene: 5ug/plate

Remarks:

With S9

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 ug/plate

Remarks:

With S9 mix

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 ug/plate

Remarks:

Without S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 ug/plate

Remarks:

Without S9 mix

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine:80 ug/plate

Remarks:

Without S9 mix

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide: 0.2 ug/plate

Remarks:

Without S9 mix

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 ug/plate

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for reductions in bacterial lawn in both experiment 1 and 2.

Evaluation criteria:

Acceptance Criteria

The reverse mutation assay may be considered valid if the following criteria are met:

All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks.

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.

All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.

Positive control chemicals must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

Evaluation Criteria

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response.

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

None

Results and discussion

Test results

Test results 1

Species / strain:

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

Remarks:

(Tested up to maximum recommended dose of 5000 ug/plate)

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:

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

Remarks:

(Tested up to maximum recommended dose of 5000 ug/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Test Results for Each Experiment are as follows:

Codes:

 

ENNG     N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO      4-Nitroquinoline-1-oxide

9AA         9-Aminoacridine

I               Intense test item induced coloration

#               Standard deviation

BP            Benzo(a)pyrene

2AA      2-Aminoanthracene

 

Table 1          Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 03 December 2013					To: 06 December 2013
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	115 108 79	(101) 19.1#	12 20 25	(19) 6.6	23 15 24		(21) 4.9	28 25 13	(22) 7.9	7 8 15	(10) 4.4
	1.5 µg	87 114 99	(100) 13.5	17 16 20	(18) 2.1	27 17 17		(20) 5.8	28 16 20	(21) 6.1	12 17 13	(14) 2.6
	5 µg	76 86 102	(88) 13.1	13 9 28	(17) 10.0	12 12 12		(12) 0.0	27 27 21	(25) 3.5	13 17 9	(13) 4.0
	15 µg	86 99 95	(93) 6.7	21 13 28	(21) 7.5	13 15 23		(17) 5.3	24 20 19	(21) 2.6	11 19 12	(14) 4.4
	50 µg	84 102 94	(93) 9.0	17 21 13	(17) 4.0	20 15 23		(19) 4.0	13 16 28	(19) 7.9	5 11 13	(10) 4.2
	150 µg	115 107 96	(106) 9.5	15 20 17	(17) 2.5	13 17 13		(14) 2.3	20 19 19	(19) 0.6	11 12 17	(13) 3.2
	500 µg	120 96 100	(105) 12.9	16 19 13	(16) 3.0	27 25 15		(22) 6.4	25 11 24	(20) 7.8	8 8 17	(11) 5.2
	1500 µg	100 100 127	(109) 15.6	21 24 11	(19) 6.8	12 12 13		(12) 0.6	19 13 15	(16) 3.1	9 4 12	(8) 4.0
	5000 µg	103 75 74	(84) 16.5	11 20 12	(14) 4.9	16 16 20		(17) 2.3	24 25 15	(21) 5.5	7 5 9	(7) 2.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		421 472 425	(439) 28.4	131 187 163	(160) 28.1	337 425 299		(354) 64.6	183 134 152	(156) 24.8	567 581 496	(548) 45.6

 

 

Table 2         Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 03 December 2013					To: 06 December 2013
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	104 83 82	(90) 12.4#	16 11 8	(12) 4.0	21 17 19		(19) 2.0	23 25 17	(22) 4.2	5 16 11	(11) 5.5
	1.5 µg	94 99 87	(93) 6.0	8 13 15	(12) 3.6	17 20 13		(17) 3.5	27 23 27	(26) 2.3	9 19 11	(13) 5.3
	5 µg	86 104 111	(100) 12.9	13 16 12	(14) 2.1	15 23 9		(16) 7.0	23 20 17	(20) 3.0	11 13 16	(13) 2.5
	15 µg	92 91 87	(90) 2.6	15 11 12	(13) 2.1	13 15 17		(15) 2.0	19 17 20	(19) 1.5	7 12 13	(11) 3.2
	50 µg	79 120 114	(104) 22.1	11 13 12	(12) 1.0	11 17 24		(17) 6.5	23 19 17	(20) 3.1	9 8 11	(9) 1.5
	150 µg	98 84 95	(92) 7.4	15 11 12	(13) 2.1	20 20 24		(21) 2.3	12 24 20	(19) 6.1	9 15 12	(12) 3.0
	500 µg	90 102 111	(101) 10.5	21 12 16	(16) 4.5	21 21 20		(21) 0.6	9 21 21	(17) 6.9	11 12 15	(13) 2.1
	1500 µg	91 98 102	(97) 5.6	17 11 8	(12) 4.6	24 20 20		(21) 2.3	16 19 19	(18) 1.7	16 17 13	(15) 2.1
	5000 µg	98 102 98	(99) 2.3	11 9 17	(12) 4.2	19 24 13		(19) 5.5	19 21 16	(19) 2.5	12 4 16	(11) 6.1
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1244 776 877	(966) 246.3	285 222 215	(241) 38.6	294 270 290		(285) 12.9	222 241 266	(243) 22.1	241 452 452	(382) 121.8

 

 

Table 3          Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 16 December 2013					To: 19 December 2013
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	80 66 67	(71) 7.8#	14 17 16	(16) 1.5	11 17 12		(13) 3.2	14 28 14	(19) 8.1	11 4 11	(9) 4.0
	50 µg	68 90 94	(84) 14.0	17 16 16	(16) 0.6	11 16 15		(14) 2.6	15 19 16	(17) 2.1	12 7 7	(9) 2.9
	150 µg	85 64 73	(74) 10.5	12 14 15	(14) 1.5	11 16 11		(13) 2.9	12 9 19	(13) 5.1	6 8 5	(6) 1.5
	500 µg	78 68 76	(74) 5.3	12 17 20	(16) 4.0	12 12 18		(14) 3.5	10 21 9	(13) 6.7	14 8 9	(10) 3.2
	1500 µg	85 72 62	(73) 11.5	20 15 20	(18) 2.9	14 10 13		(12) 2.1	10 14 13	(12) 2.1	8 8 7	(8) 0.6
	5000 µg	67 67 66	(67) 0.6	6 10 10	(9) 2.3	18 15 15		(16) 1.7	18 19 14	(17) 2.6	12 6 7	(8) 3.2
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		636 595 624	(618) 21.1	770 776 1336	(961) 325.1	464 431 386		(427) 39.2	181 195 198	(191) 9.1	704 437 1086	(742) 326.2

 

 

Table 4          Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 16 December 2013					To: 19 December 2013
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	81 65 76	(74) 8.2#	11 11 11	(11) 0.0	19 18 19		(19) 0.6	14 17 20	(17) 3.0	8 8 8	(8) 0.0
	50 µg	71 80 76	(76) 4.5	7 9 9	(8) 1.2	19 18 22		(20) 2.1	29 19 24	(24) 5.0	7 9 15	(10) 4.2
	150 µg	79 68 88	(78) 10.0	5 7 9	(7) 2.0	22 24 15		(20) 4.7	27 22 14	(21) 6.6	12 8 13	(11) 2.6
	500 µg	73 79 61	(71) 9.2	10 7 10	(9) 1.7	14 11 25		(17) 7.4	11 19 19	(16) 4.6	9 11 9	(10) 1.2
	1500 µg	92 74 93	(86) 10.7	8 8 13	(10) 2.9	20 21 18		(20) 1.5	22 17 24	(21) 3.6	11 9 10	(10) 1.0
	5000 µg	87 82 81	(83) 3.2	9 4 7	(7) 2.5	21 17 19		(19) 2.0	25 25 17	(22) 4.6	12 6 12	(10) 3.5
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		932 1001 1104	(1012) 86.6	201 189 205	(198) 8.3	134 110 141		(128) 16.3	119 128 107	(118) 10.5	233 197 229	(220) 19.7

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test item was tested using a protocol designed to be compatible with OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test" and Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre‑incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the plate incorporation experiment was 1.5 to 5000 µg/plate and for the pre-incubation experiment 50 to 5000 µg/plate.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion

The test item was considered to be non-mutagenic under the conditions of this test.