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EC number: 204-445-3 | CAS number: 121-03-9
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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:
- 2 010
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- 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
- 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-nitrobenzene-1-sulfonic 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
Constituent 1
Method
Species / strainopen allclose all
- 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:
- 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 1sttrial (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 1sttrail (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 2ndtrial (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 2ndtrial (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
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