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EC number: 212-298-1 | CAS number: 778-94-9
- Life Cycle description
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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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:
- 04 - 11 Mar 2019
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 26 Jun 2020
- Deviations:
- yes
- Remarks:
- Pre-test data for solvent and cytotoxicity testing not included in study report.
- GLP compliance:
- yes
- Remarks:
- The test complied with the Principles of Good Laboratory Practices (GLP) of the Certification and Accreditation Administration of the People’s Republic of China (2013 revised edition).
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-nitro-4-(trifluoromethyl)benzonitrile
- EC Number:
- 212-298-1
- EC Name:
- 2-nitro-4-(trifluoromethyl)benzonitrile
- Cas Number:
- 778-94-9
- Molecular formula:
- C8H3F3N2O2
- IUPAC Name:
- 2-nitro-4-(trifluoromethyl)benzonitrile
Constituent 1
Method
- Target gene:
- his operon
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 97a
- Additional strain / cell type characteristics:
- other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- concentration or volume of S9 mix and S9 in the final culture medium: 10% - Test concentrations with justification for top dose:
- 0.5, 1.581, 5, 15.81 and 50 µg/plate for all strains with and without metabolic activation.
The doses were selected based on the results of a pre-test with doses between 10 and 5000 µg/plate, in which bacterial toxicity was observed in all apart from the lowest concentration tested. Furthermore, slight precipitation was noted at 5000 µg/plate (-S9 conditions) only prior to the start of culturing. No precipitation occurred at any other dose group. - Vehicle / solvent:
- Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was used as solvent in the pre-test, therefore, DMSO was also selected as solvent for the main experiment.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other:
- Remarks:
- all positive controls dissolved in DMSO
1) TA97a, TA98, TA102: 50 µg/plate (-S9)
2) TA97a, TA98, TA100: 20 µg/plate (+S9)
3) TA102: 50 µg/plate (+S9)
4) TA1535: 2 µg/plate (+S9)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
3 plates for all dose groups and control groups in 2 independent experiments.
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h (first experiment), 64 h (second experiment)
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate - Rationale for test conditions:
- According to "Chemical Registration Centre of the Ministry of Environmental Protection: Chemical Test Methods – Health Effects Volume – 471 Bacterial Reverse Mutation Test [M]. Second edition. Beijing: China Environment Press, 2013: 410-416".
- Evaluation criteria:
- In a metabolically activated or not metabolically activated system, when the revertant bacterial strain count in the test sample group compared to the solvent control group exceeded a certain numerical range (that is, in the test bacteria TA1535 test sample group, the revertant bacterial strain count was equal to or exceeded the mean of the solvent control group by a factor of 3; in the other test bacteria test sample groups, the revertant bacterial strain count was equal to or exceeded the solvent control group by a factor of 2), and a dose-response relationship existed among all doses, or at a certain test point (bacterial strain or dose) the increase in revertant bacterial colony count was reproducible, then the test results could be judged positive. Otherwise, the test results were negative.
- Statistics:
- Mean values and standard deviation were calculated.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): ≥ 15.18 µg/plate S9(+): 50 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): 50 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): 50 µg/plate S9(+): 50 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): ≥ 15.18 µg/plate, S9(+): 50 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- S9(-): 15.18 µg/plate S9 (+): 50 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed
RANGE-FINDING/SCREENING STUDIES
In the pre-test, DMSO was used as the solvent. A total of seven doses were established: 5000, 1000, 300, 100, 60, 30, and 10 μg/plate. Under not metabolically activated conditions, the plate incorporation test was carried out on the test bacterial strains TA97a, TA98, TA100, TA102, and TA1535, two plates per group, and a simultaneous solvent control was established. The results of the pre-test revealed that under not metabolically activated conditions, at 5000 μg/plate dose prior to the start of culturing, there was slight precipitation, but after the end of culturing, there was no precipitation. There were no signs of precipitation for the other dose groups. For the 5000, 1000, and 300 μg/plate doses, bacterial colonies died and there was high bacterial toxicity. For the 100 and 60 μg/plate doses, bacterial colonies died or were notably reduced and there was high toxicity or moderate toxicity. For bacterial strains TA98 and TA1535 at the 30 μg/plate dose, bacterial colonies were notably reduced and there was bacterial toxicity. For the 10 μg/plate dose, there was no obvious bacterial toxicity.
STUDY RESULTS
No indication of genotoxicity was observed in any bacterial strain. For details, please refer to Tables 1 - 6 under section "Any other information on results incl. tables".
HISTORICAL CONTROL DATA: not reported - Remarks on result:
- other: not applicable
Any other information on results incl. tables
Table 1: Results of the first experiment (individual values)
μg/plate | No. | S9- | S9+ | ||||||||
TA97a | TA98 | TA100 | TA102 | TA1535 | TA97a | TA98 | TA100 | TA102 | TA1535 | ||
Solvent control | 1 | 183 | 13 | 108 | 310 | 14 | 197 | 24 | 119 | 274 | 19 |
2 | 165 | 16 | 106 | 296 | 8 | 199 | 22 | 121 | 259 | 20 | |
3 | 158 | 16 | 124 | 316 | 8 | 186 | 27 | 143 | 265 | 24 | |
50 | 1 | 51 | 2 | 0 | 256 | 0 | 180 | 8 | 32 | 211 | 0 |
2 | 49 | 5 | 0 | 241 | 0 | 170 | 17 | 26 | 207 | 0 | |
3 | 58 | 3 | 0 | 220 | 0 | 179 | 16 | 33 | 223 | 0 | |
15.81 | 1 | 201 | 18 | 73 | 260 | 3 | 211 | 18 | 168 | 282 | 15 |
2 | 194 | 16 | 62 | 274 | 4 | 195 | 14 | 153 | 261 | 13 | |
3 | 211 | 20 | 85 | 225 | 6 | 178 | 22 | 134 | 258 | 16 | |
5 | 1 | 147 | 14 | 102 | 320 | 9 | 155 | 23 | 123 | 257 | 16 |
2 | 131 | 18 | 103 | 357 | 6 | 100 | 18 | 125 | 275 | 12 | |
3 | 193 | 14 | 90 | 297 | 8 | 179 | 17 | 143 | 257 | 16 | |
1.581 | 1 | 161 | 20 | 125 | 286 | 12 | 149 | 18 | 124 | 201 | 10 |
2 | 154 | 27 | 101 | 344 | 6 | 154 | 19 | 146 | 222 | 22 | |
3 | 145 | 12 | 103 | 314 | 13 | 139 | 22 | 138 | 243 | 17 | |
0.5 | 1 | 176 | 26 | 98 | 293 | 15 | 233 | 25 | 157 | 266 | 19 |
2 | 185 | 19 | 151 | 298 | 10 | 232 | 15 | 156 | 257 | 16 | |
3 | 150 | 18 | 80 | 296 | 12 | 213 | 28 | 165 | 281 | 25 | |
Positive control | 1 | 3200 | 1132 | 882 | 1346 | 229 | 1128 | 846 | 1026 | 986 | 326 |
2 | 2974 | 1008 | 768 | 1228 | 227 | 1360 | 886 | 1148 | 942 | 348 | |
3 | 2624 | 1184 | 776 | 1264 | 253 | 1208 | 986 | 1266 | 884 | 326 |
Table 2: Results of the first experiment without metabolic activation (mean values)
μg/plate | Statistical results | S9- | ||||
TA97a | TA98 | TA100 | TA102 | TA1535 | ||
Solvent control | Mean ± SD Ratio | 168.67 ± 12.90 1.00 | 15.00 ± 1.73 1.00 | 112.67 ± 9.87 1.00 | 307.33 ± 10.26 1.00 | 10.00 ± 3.46 1.00 |
50 | Mean ± SD Ratio | 52.67 ± 4.73 0.31 | 3.33 ± 1.53 0.22 | 0.00 ± 0.00 0.00 | 239.00 ± 18.08 0.78 | 0.00 ± 0.00 0.00 |
15.81 | Mean ± SD Ratio | 202.00 ± 8.54 1.20 | 18.00 ± 2.00 1.20 | 73.33 ± 11.50 0.65 | 253.00 ± 25.24 0.82 | 4.33 ± 1.53 0.43 |
5 | Mean ± SD Ratio | 157.00 ± 32.19 0.93 | 15.33 ± 2.31 1.02 | 98.33 ± 7.23 0.87 | 324.67 ± 30.27 1.06 | 7.67 ± 1.53 0.77 |
1.581 | Mean ± SD Ratio | 153.33 ± 8.02 0.91 | 19.67 ± 7.51 1.31 | 109.67 ± 13.32 0.97 | 314.67 ± 29.01 1.02 | 10.33 ± 3.79 1.03 |
0.5 | Mean ± SD Ratio | 170.33 ± 18.18 1.01 | 21.00 ± 4.36 1.40 | 109.67 ± 36.91 0.97 | 295.67 ± 2.52 0.96 | 12.33 ± 2.52 1.23 |
Positive control | Mean ± SD Ratio | 2932.67 ± 290.22 17.39 | 1108.00 ± 90.42 73.87 | 808.67 ± 63.63 7.18 | 1279.33 ± 60.48 4.16 | 236.33 ± 14.47 23.63 |
Table 3: Results of the first experiment with metabolic activation (mean values)
μg/plate | Statistical results | S9+ | ||||
TA97a | TA98 | TA100 | TA102 | TA1535 | ||
Negative control | Mean ± SD Ratio | 194.00 ± 7.00 1.00 | 24.33 ± 2.52 1.00 | 127.67 ± 13.32 1.00 | 266.00 ± 7.55 1.00 | 21.00 ± 2.65 1.00 |
50 | Mean ± SD Ratio | 176.33 ± 5.51 0.91 | 13.67 ± 4.93 0.56 | 30.33 ± 3.79 0.24 | 213.67 ± 8.33 0.80 | 0.00 ± 0.00 0.00 |
15.81 | Mean ± SD Ratio | 194.67 ± 16.50 1.00 | 18.00 ± 4.00 0.74 | 151.67 ± 17.04 1.19 | 267.00 ± 13.08 1.00 | 14.67 ± 1.53 0.70 |
5 | Mean ± SD Ratio | 144.67 ± 40.50 0.75 | 19.33 ± 3.21 0.79 | 130.33 ± 11.02 1.02 | 263.00 ± 10.39 0.99 | 14.67 ± 2.31 0.70 |
1.581 | Mean ± SD Ratio | 147.33 ± 7.64 0.76 | 19.67 ± 2.08 0.81 | 136.00 ± 11.14 1.07 | 222.00 ± 21.00 0.83 | 16.33 ± 6.03 0.78 |
0.5 | Mean ± SD Ratio | 226.00 ± 11.27 1.16 | 22.67 ± 6.81 0.93 | 159.33 ± 4.93 1.25 | 268.00 ± 12.12 1.01 | 20.00 ± 4.58 0.95 |
Positive control | Mean ± SD Ratio | 1232.00 ± 117.85 6.35 | 906.00 ± 72.11 37.23 | 1146.67 ± 120.01 8.98 | 937.33 ± 51.16 3.52 | 333.33 ± 12.70 15.87 |
Table 4: Results of the second experiment (individual values)
μg/plate | No. | S9- | S9+ | ||||||||
TA97a | TA98 | TA100 | TA102 | TA1535 | TA97a | TA98 | TA100 | TA102 | TA1535 | ||
Solvent control | 1 | 212 | 23 | 139 | 248 | 21 | 207 | 28 | 155 | 345 | 36 |
2 | 196 | 29 | 104 | 278 | 34 | 205 | 28 | 157 | 314 | 29 | |
3 | 191 | 23 | 127 | 295 | 30 | 192 | 24 | 143 | 327 | 24 | |
50 | 1 | 60 | 4 | 0 | 198 | 0 | 277 | 15 | 77 | 194 | 3 |
2 | 57 | 3 | 0 | 194 | 0 | 245 | 20 | 97 | 203 | 4 | |
3 | 40 | 3 | 0 | 224 | 0 | 288 | 17 | 100 | 212 | 6 | |
15.81 | 1 | 247 | 21 | 94 | 216 | 5 | 302 | 21 | 199 | 260 | 30 |
2 | 204 | 28 | 87 | 265 | 9 | 297 | 17 | 205 | 257 | 27 | |
3 | 201 | 27 | 105 | 222 | 24 | 245 | 24 | 182 | 249 | 29 | |
5 | 1 | 189 | 19 | 147 | 263 | 24 | 291 | 27 | 137 | 300 | 28 |
2 | 184 | 23 | 144 | 222 | 25 | 278 | 28 | 137 | 266 | 27 | |
3 | 196 | 31 | 135 | 236 | 16 | 263 | 31 | 142 | 278 | 32 | |
1.581 | 1 | 199 | 19 | 96 | 237 | 29 | 273 | 23 | 118 | 250 | 28 |
2 | 169 | 23 | 143 | 252 | 22 | 248 | 22 | 151 | 231 | 45 | |
3 | 181 | 20 | 114 | 244 | 16 | 232 | 25 | 145 | 268 | 32 | |
0.5 | 1 | 171 | 24 | 136 | 281 | 20 | 220 | 34 | 214 | 266 | 43 |
2 | 164 | 30 | 103 | 257 | 27 | 218 | 30 | 201 | 282 | 30 | |
3 | 209 | 19 | 98 | 260 | 25 | 209 | 29 | 222 | 270 | 29 | |
Positive control | 1 | 3488 | 872 | 606 | 927 | 364 | 1102 | 678 | 789 | 1128 | 223 |
2 | 3168 | 1064 | 768 | 836 | 403 | 987 | 658 | 897 | 1287 | 307 | |
3 | 3296 | 928 | 559 | 1024 | 375 | 1036 | 712 | 778 | 996 | 287 |
Table 5: Results of the second experiment without metabolic activation (mean values)
μg/plate | Statistical results | S9- | ||||
TA97a | TA98 | TA100 | TA102 | TA1535 | ||
Negative control | Mean ± SD Ratio | 199.67 ± 10.97 1.00 | 25.00 ± 3.46 1.00 | 123.33 ± 17.79 1.00 | 273.67 ± 23.80 1.00 | 28.33 ± 6.66 1.00 |
50 | Mean ± SD Ratio | 52.33 ± 10.79 0.26 | 3.33 ± 0.58 0.13 | 0.00 ± 0.00 0.00 | 205.33 ± 16.29 0.75 | 0.00 ± 0.00 0.00 |
15.81 | Mean ± SD Ratio | 217.33 ± 25.74 1.09 | 25.33 ± 3.79 1.01 | 95.33 ± 9.07 0.77 | 234.33 ± 26.73 0.86 | 12.67 ± 10.02 0.45 |
5 | Mean ± SD Ratio | 189.67 ± 6.03 0.95 | 24.33 ± 6.11 0.97 | 142.00 ± 6.24 1.15 | 240.33 ± 20.84 0.88 | 21.67 ± 4.93 0.76 |
1.581 | Mean ± SD Ratio | 183.00 ± 15.10 0.92 | 20.67 ± 2.08 0.83 | 117.67 ± 23.71 0.95 | 244.33 ± 7.51 0.89 | 22.33 ± 6.51 0.79 |
0.5 | Mean ± SD Ratio | 181.33 ± 24.21 0.91 | 24.33 ± 5.51 0.97 | 112.33 ± 20.65 0.91 | 266.00± 13.08 0.97 | 24.00 ± 3.61 0.85 |
Positive control | Mean ± SD Ratio | 3317.33 ± 161.06 16.61 | 954.67 ± 98.74 38.19 | 644.33 ± 109.65 5.22 | 929.00 ± 94.02 3.39 | 380.67 ± 20.11 13.44 |
Table 6: Results of the second experiment with metabolic activation (mean values)
μg/plate | Statistical results | S9+ | ||||
TA97a | TA98 | TA100 | TA102 | TA1535 | ||
Negative control | Mean ± SD Ratio | 201.33 ± 8.14 1.00 | 26.67 ± 2.31 1.00 | 151.67 ± 7.57 1.00 | 328.67 ± 15.57 1.00 | 29.67 ± 6.03 1.00 |
50 | Mean ± SD Ratio | 270.00 ± 22.34 1.34 | 17.33± 2.52 0.65 | 91.33 ± 12.50 0.60 | 203.00 ± 9.00 0.62 | 4.33 ± 1.53 0.15 |
15.81 | Mean ± SD Ratio | 281.33 ± 31.56 1.40 | 20.67 ± 3.51 0.78 | 195.33 ± 11.93 1.29 | 255.33 ± 5.69 0.78 | 28.67 ± 1.53 0.97 |
5 | Mean ± SD Ratio | 277.33 ± 14.01 1.38 | 28.67 ± 2.08 1.08 | 138.67 ± 2.89 0.91 | 281.33 ± 17.24 0.86 | 29.00 ± 2.65 0.98 |
1.581 | Mean ± SD Ratio | 251.00 ± 20.66 1.25 | 23.33 ± 1.53 0.88 | 138.00 ± 17.58 0.91 | 249.67 ± 18.50 0.76 | 35.00 ± 8.89 1.18 |
0.5 | Mean ± SD Ratio | 215.67 ± 5.86 1.07 | 31.00 ± 2.65 1.16 | 212.33 ± 10.60 1.40 | 272.67 ± 8.33 0.83 | 34.00 ± 7.81 1.15 |
Positive control | Mean ± SD Ratio | 1041.67 ± 57.71 5.17 | 682.67 ± 27.30 25.60 | 821.33 ± 65.76 5.42 | 1137.00 ± 145.71 3.46 | 272.33 ± 43.88 9.18 |
Applicant's summary and conclusion
- Conclusions:
- The test results indicated that, under the conditions of this test, 2-nitro-4-(trifluoromethyl)benzonitrile did not have mutagenicity on the test bacteria.
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