Toluene-2-sulphonamide

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:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Toluene-2-sulphonamide

EC Number:

201-808-8

EC Name:

Toluene-2-sulphonamide

Cas Number:

88-19-7

Molecular formula:

C7H9NO2S

IUPAC Name:

2-methylbenzene-1-sulfonamide

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Public Health (obtained December 19, 1994)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and 5,6-benzoflavone (BF) induced rat liver S9.

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 100 - 5000 μg/plate; no bacterial toxicity up to highest dose level.
- Main tests 1 and 2: 0, 312.5, 625, 1250, 2500 and 5000 μg / plate.
- Justification: In order to determine the appropriate concentration of the test substance in this test, tests were conducted by the same experimental method as this test using six concentrations of 100, 200, 500, 1000, 2000 and 5000 μg/plate for all indicator strains. The test was performed on one plate of each concentration. As a result, regardless of the presence or absence of metabolic activation, no growth inhibition was observed at any concentration in any strain.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was reported to be soluble in DMSO, slightly soluble in water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene (2-AA)

Remarks:

see table 4 in 'Any other information on materials and methods'

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Plate incorporation method, with and without metabolic activation. 0.1 mL of the solvent, test item solution or positive control substance solution to were put in a test tube; then, 0.5 ml of phosphate buffer solution (or 0.5 ml of S9 mixture solution in metabolic activation test) and 0.1 ml of assay bacterial solution were mixed in, and cultured with shaking at 37ºC for 20 minutes. Next, a mixture containing 2 ml of top agar (kept at 45ºC) was overlaid on the plate. After solidification, plates were incubated at 37ºC for 48 hours. After the incubation period, the number of revertant colonies was counted, and at the same time, the presence or absence of growth inhibition of the indicator strain was observed using a stereomicroscope.
- Cell density at seeding (if applicable): see table 3.

DURATION
- Preincubation period: no.
- Exposure duration: 48h

SELECTION AGENT (mutation assays): No specific selection agent was used. The lack of amino-acid in the medium allowed only mutants to grow because of their ability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.

Evaluation criteria:

The results were judged as positive if the following three criteria were satisfied based on the average number of revertant colonies on the plate at each concentration.
1) In the test substance treatment group, the number of revertant colonies was increased more than twice the solvent control value.
2) The number of revertant colonies increases with increasing test substance concentration (concentration dependence).
3) The repeatability of the increase in the number of revertant colonies is observed from the results of the two tests.

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 applicable

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:

not applicable

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:

not applicable

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:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: none reported

RANGE-FINDING/SCREENING STUDIES: in the preliminary test, performed in the range of 100 to 5000 μg/plate, no growth inhibition by the test substance was observed in any of the strains regardless of the presence or absence of metabolic activation. Therefore, the top dose for the main tests was 5000 μg/plate, and the next doses were 2500, 1250, 625 and 312.5 μg/plate (common ratio 2).

HISTORICAL CONTROL DATA: not specified.

Any other information on results incl. tables

Table 1. Results of reverse mutation test of o-TSA on bacteria (1st trial) without metabolic activation (direct method).

Test substance concentration (µg/plate)	Number of revertant colonies per plate (Mean ± S.D.)
	TA100			TA1535			WP2 uvrA			TA98			TA1537
0	124	136	126	8	8	7	10	17	7	16	19	24	9	11	14
	(129 ± 6)			(8 ± 1)			(11 ± 5)			(20 ± 4)			(11 ± 3)
312.5	116	127	145	7	8	18	15	19	13	23	15	26	12	13	8
	(129 ± 15)			(11 ± 6)			(16 ± 3)			(21 ± 6)			(11 ± 3)
625	144	138	105	9	6	9	10	13	14	21	22	24	10	3	10
	(129 ± 21)			(8 ± 2)			(12 ± 2)			(22 ± 7)			(11 ± 2)
1250	137	121	103	10	5	10	18	16	6	13	17	19	7	10	7
	(120 ± 17)			(8 ± 3)			(13 ± 6)			(16 ± 3)			(8 ± 2)
2500	114	125	101	11	7	7	12	14	18	21	21	22	5	9	8
	(113 ± 12)			(8 ± 2)			(15 ± 3)			(21 ± 1)			(7 ± 2)
5000	119	111	130	7	8	7	15	14	10	17	22	15	14	7	11
	(120 ± 10)			(7 ± 1)			(13 ± 3)			(18 ± 4)			(11 ± 4)
Posit. control	803	847	873	340	313	321	1125	1177	1106	292	298	308	978	906	641
	(841 ± 35)			(325 ± 14)			(1136 ± 37)			(299 ± 8)			(842 ± 177)

Table 2. Results of reverse mutation test of o-TSA on bacteria (1st trial) with metabolic activation (S9 mix).

Test substance concentration (µg/plate)	Number of revertant colonies per plate (Mean ± S.D.)
	TA100			TA1535			WP2 uvrA			TA98			TA1537
0	120	152	123	8	11	8	11	20	25	32	46	37	13	10	13
	(132 ± 18)			(0 ± 2)			(19 ± 7)			(38 ± 7)			(12 ± 2)
312.5	154	128	138	11	10	10	15	8	19	37	34	37	15	8	0
	(140 ± 13)			(10 ± 1)			(14 ± 6)			(36 ± 2)			(11 ± 4)
625	136	129	142	13	11	11	14	17	21	51	45	44	14	18	0
	(145 ± 11)			(9 ± 2)			(13 ± 2)			(41 ± 7)			(13 ± 4)
1250	157	135	143	8	11	8	15	11	13	35	49	38	11	10	17
	(132 ± 11)			(11 ± 4)			(17 ± 5)			(37 ± 7)			(16 ± 4)
2500	139	137	119	8	15	11	21	17	38	30	44		16	13	20
	(132 ± 11)			(11 ± 4)			(17 ± 5)			(37 ± 7)			(16 ± 4)
5000	120	130	126	16	12	9	16	18	12	36	43	37	18	14	19
	(125 ± 5)			(12 ± 4)			(15 ± 3)			(39 ± 4)			(17 ± 3)
Posit. control	548	589	604	127	128	120	331	1251	1307	324	337	316	108	94	105
	(580 ± 29)			(125 ± 4)			(1297 ± 42)			(326 ± 11)			(102 ± 7)

Table 3. Results of reverse mutation test of o-TSA on bacteria (2nd trial) without metabolic activation (direct method).

Test substance concentration (µg/plate)				Number of revertant colonies per plate (Mean ± S.D.)
	TA100			TA1535			WP2 uvrA							TA9S							TA1537
0	117	134	114	10	10	11	9	13			11			16	20			22			7	6			3
	(122 ± 11)			(10 ± 1)			(11 ± 2)							(19 ± 3)							( 5 ± 2)
312.5	139	119	139	6	10	9	23	14			14			31	20			28			5	6			3
	(132 ± 12)			( S ± 2)			(17 ± 5)							(26 ± 6)							( 5 ± 2)
625	107	121	126	6	7	7	14	10			11			27	14			19			7	8			3
	(118 ± 10)			( 7 ± 1)			(12 ± 2)							(20 ± 7)							( 6 ± 3)
1250	105	107	103	10	6	6	15			S			9	13			20		19		7			5			7
	(105 ± 2)			( 7 ± 2)			(11 ± 4)							(17 ± 4)							( 6 ± 1)
2500	125	93	100	12	4	12	19		12			14		20		19				14	3		6			12
	(103 ± 17)			( 9 ± 5)			(15 ± 4)							(18 ± 3)							( 7 ± 5)
5000	102	91	101	7	7	6	15	12			16			17	16			19			5	8			5
	( 99 ± 4)			( 7 ± 1)			(14 ± 2)							(17 ± 2)							( 6 ± 2)
Positive control	797	793	795	301	311	293	961	1109			993			264	262			305			606	654			610
	(795 ± 2)			(303 ± 9)			(1028 ± 71)							(277 ± 2-1)							(621 ± 26)

Table 4. Results of reverse mutation test of o-TSA on bacteria (2nd trial) with metabolic activation (S9 mix).

Test substance concentration (µg/plate)				Number of revertant colonies per plate (Mean ± S.D.)
	TA100			TA1535			WP2 uvrA							TA9S			TA1537
0	154	144	136	8	6	15	20	20			25			35	42	39	11	13	9
	(145 ± 9)			(10 ± 5)			(22 ± 3)							(39 ± 4)			(11 ± 2)
312.5	165	157	156	7	11	10	22	20			13			31	41	41	6	7	9
	(159 ± 5)			(9 ± 2)			(18 ± 5)							(38 ± 6)			(7 ± 2)
625	157	155	144	13	9	18	12	23			11			37	37	41	13	10	8
	(152 ± 7)			(13 ± 5)			(15 ± 7)							(38 ± 2)			(10 ± 3)
1250	158	131	120	12	7	13	23			16			10	42	30	43	9	12	10
	(136 ± 20)			(11 ± 3)			(16 ± 7)							(38 ± 7)			(10 ± 2)
2500	122	111	123	11	7	7	17		18			11		46	39	43	12	7	17
	(119 ± 7)			(8 ± 2)			(15 ± 4)							(43 ± 3)			(12 ± 5)
5000	134	125	110	8	8	14	10	13			18			38	27	32	15	10	8
	( 123 ± 12)			(10 ± 3)			(14 ± 4)							(32 ± 6)			(11 ± 4)
Positive control	504	499	472	127	160	132	1207	1170			1290			357	373	341	106	136	101
	(492 ± 17)			(140 ± 18)			(1222 ± 61)							(357 ± 16)			(114 ± 19)

 

Applicant's summary and conclusion

Conclusions:

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. Therefore, the test item was not mutagenic under test conditions.

Executive summary:

The ability of the test item to induce mutation was studied in a bacterial reverse mutation test (Ames test) according to OECD Guidelines No. 471 and the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and tryptophan requiring Escherichia coli WP2 uvrA were tested in triplicate at six concentrations of test item in up to 5000 μg/plate, with and without metabolic activation (S9 mix), based on the results of a preliminary range-finding test. The plate incorporation method was used. Vehicle (DMSO) and positive controls were run in parallel. No cytotoxicity was observed at any dose tested, the vehicle control plates gave counts of revertant colonies within the normal range, and 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. All validity criteria were met. 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. Therefore, the test item was not mutagenic under test conditions.