4-nitrotoluene-2-sulphonic acid

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene-2-sulphonic acid

EC Number:

204-445-3

EC Name:

4-nitrotoluene-2-sulphonic acid

Cas Number:

121-03-9

Molecular formula:

C7H7NO5S

IUPAC Name:

2-methyl-5-nitrobenzenesulfonic acid

Details on test material:

- Name of test material (as cited in study report): 2-methyl-5-nitrobenzene sulfonic acid
- Molecular formula (if other than submission substance): C7H7NO5S
- Molecular weight (if other than submission substance): 217.20
- Physical state: liquid
- Analytical purity: 79.60 %
- Impurities (identity and concentrations): 15.90% water, 1.75% NaSO4, 0.01% NaCl, 2.74% sulfonic acid
- Lot/batch No.: 96001

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from sprague Dawlay rat livers

Test concentrations with justification for top dose:

0, 158, 313, 625, 1250, 2500, 5000 µg/plate (all strains with/without activation)
0, 1000, 2000, 3000, 4000, 5000 µg/plate (TA1537, confirmative examination; with/without activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Controls

Positive controls:

yes

Positive control substance:

other: see details

Details on test system and experimental conditions:

Tests were conducted both groups with and without S9 mix through pre-incubation method which has been developed from the original method devised by Ames et al.
100μL solution of the solvent, the test substance or the positive control substance and then in the groups without S9 mix, 500μL of 0.1M sodium phosphate buffer solution (pH 7.4) were poured, and in the groups with S9 mix, 500μ L of S9 mix and 100 µL liquid of the strain were poured into a test tube, followed by shake culture at 37˚C for 20 minutes (pre-incubation). After the pre-incubation, 2 mL top agar was added to the mixed liquid, which was then layered onto the plate.
After each plate was cultured at 37˚C for 48 hours, using a stereo-microscope (x60), growing conditions of the tester strains on the plate were observed to examine the growth inhibiting effect of the test substance on the tester strains. Then the number of reverse mutated colonies on each plate was counted by a colony analyzer (CA-11: System Science Ltd.). Tests were independently conducted twice.


Positive Control:
They were dissolved with DMSO and freeze-stored (at -20˚C) after being injected each by a small amount.
without S9-mix
2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2: Wako Pure Chemical Industries, Ltd.) (TA100, TA98 and WP" uvrA)
Sodium azide (NaN3: Wako Pure Chemical Industries, Ltd.) (TA1535)
9-Aminoacridine hydrochloride (ACR: ALDRICH Ltd.) (TA1537)
with S9-mix
2-aminoanthracene (2-AA: Wako Pure Chemical Industries, Ltd.) (all strains)

Evaluation criteria:

If the number of reverse mutated colonies (mean value) on a plate is twice or more, compared to that for the solvent control, and if reproducibility and dose-dependence were also recognized, the test substance was determined to be positive.

Statistics:

Verification using a statistical method was not performed.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

Any growth inhibition effects by 2-methyl-5-nitrobenzenesulfonic acid were not observed in the groups both with and without S9 mix. A dose-dependent tendency in the increase of mutated colonies was observed in the TA100, TA98 and TA1537 groups without S9 mix and in the TA100 group with S9 mix. In the trials which were repeated twice, the number of mutated colonies in the TA1535 group was 1.5 to 2 times higher than that of the solvent control, but it could not be determined to be definitely positive. A confirmative test was carried out with regard to the TA1535 strains with and without S9 mix to confirm its reproducibility, but a clear mutagenicity-inducing effect was not observed.(The specific mutagen activity showing a comparative value of the mutagen strength was 42.1). On the other hand, the positive control substance induced two times or more of mutated colonies in each tester strain, compared to the solvent control group. In addition, remarkable changes such as precipitation were not observed during the tests.
From the results mentioned above, it is concluded that 2-methy-5-nitrobenzenesulfonic acid is positive in terms of mutagenicity against micro-organisms.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 Results 1^sttrial (direct method: -S9)

Test Sub.	(µg/plate)	TA 100	TA1535	WP2 uvrA	TA 98	TA 1537
	0	112 ± 2	18 ± 1	29 ± 1	26 ± 1	7 ± 1
	156	110 ± 3	17 ± 4	31 ± 5	33 ± 5	5 ± 1
	313	105 ± 3	15 ± 2	28 ± 2	44 ± 3	5 ± 0
	625	111 ± 4	15 ± 1	32 ± 3	52 ± 3	6 ± 1
	1250	128 ± 8	12 ± 3	32 ± 6	56 ± 5	9 ± 2
	2500	150 ± 12	21 ± 3	29 ± 1	99 ± 13	15 ± 2
	5000	204 ± 8	22 ± 2	36 ± 4	145 ± 5	31 ± 4
Positive control		590 ± 15	393 ± 26	175 ± 18	455 ± 3	534 ± 4

 

Results 1^sttrail (activation method: +S9)

Test Sub.	(µg/plate)	TA 100	TA1535	WP2 uvrA	TA 98	TA 1537
	0	114 ± 6	14 ± 2	32 ± 5	32 ± 2	12 ± 2
	156	114 ± 4	20 ± 2	27 ± 1	27 ± 2	8 ± 1
	313	116 ± 12	19 ± 2	28 ± 2	30 ± 4	13 ± 2
	625	117 ± 2	15 ± 3	32 ± 4	32 ± 1	11 ± 3
	1250	128 ± 14	13 ± 2	31 ± 2	31 ± 3	11 ± 1
	2500	161 ± 2	19 ± 3	32 ± 3	36 ± 3	17 ± 1
	5000	254 ± 6	27 ± 2	34 ± 6	48 ± 4	17 ± 3
Positive control		733 ± 6	386 ± 11	413 ± 18	366 ± 16	159 ± 14

  

Results 2^ndtrial (direct method: -S9)

Test Sub.	(µg/plate)	TA 100	TA1535	WP2 uvrA	TA 98	TA 1537
	0	129 ± 5	20 ± 3	27 ± 4	26 ± 2	8 ± 1
	156	125 ± 6	21 ± 3	25 ± 0	32 ± 3	6 ± 1
	313	121 ± 3	21 v 3	27 ± 3	28 ± 2	5 ± 0
	625	126 ± 9	23 ± 3	32 ± 5	44 ± 3	5 ± 1
	1250	147 ± 9	28 ± 4	26 ± 3	50 ± 4	9 ± 2
	2500	183 ± 3	24 ± 2	31 ± 1	90 ± 8	14 ± 2
	5000	285 ± 1	41 ± 6	31 ± 3	131 ± 2	32 ± 4
Positive control		543 ± 11	455 ± 42	188 ± 11	470 ± 23	556 ± 22

 

 Results 2^ndtrial (activation method: +S9)

Test Sub.	(µg/plate)	TA 100	TA1535	WP2 uvrA	TA 98	TA 1537
	0	127 ± 3	16 ± 2	32 ± 4	29 ± 5	10 ± 2
	156	133 ± 2	17 ± 2	31 ± 2	27 ± 3	11 ± 4
	313	126 ± 11	18 ± 5	33 ± 4	29 ± 1	11 ± 1
	625	134 ± 12	18 ± 4	33 ± 6	27 ± 3	10 ± 2
	1250	147 ± 11	20 ± 2	31 ± 3	29 ± 6	8 ± 1
	2500	174 ± 11	20 ± 1	30 ± 3	31 ± 1	16 ± 1
	5000	296 ± 14	26 ± 0	31 ± 5	38 ± 4	15 ±2
Positive control		699 ± 11	376 ± 21	465 ± 6	372 ± 18	161 ± 11

 

 Results of the confirmative examination (direct method: -S9)

Test sub.	(µg/plate)	TA 1535
	0	19 ± 2
	1000	24 ± 5
	2000	18 ± 2
	3000	19 ± 1
	4000	18 ± 2
	5000	20 ± 2
Positive control		452 ± 24

 

Results of the confirmative examination (activation method: +S9)

Test sub.	(µg/plate)	TA 1535
	0	18 ± 3
	1000	17 ± 1
	2000	13 ± 1
	3000	16 ± 1
	4000	22 ± 3
	5000	20 ± 2
Positive control		338 ± 35

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous