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EC number: 201-853-3 | CAS number: 88-72-2
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:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable deviations
Data source
Referenceopen allclose all
- Reference Type:
- publication
- Title:
- Salmonella Mutagenicity Test results for 250 Chemicals
- Author:
- Haworth S, Lawlor T, Mortelmans K, Speck W, and Zeiger E
- Year:
- 1 983
- Bibliographic source:
- Environmental Mutagenesis Supplement 1:3-142
- Reference Type:
- publication
- Title:
- National Toxicology Program, NTP Technical report on toxicity studies of o-, m-, and p-Nitrotoluenes, administered in dosed feed to F344/N rats and B6C3F1 mice
- Author:
- US Department of Health and Human Services
- Year:
- 1 992
- Bibliographic source:
- NTP technical report series 23, NIH Publ.-No. 96-3346
- Reference Type:
- publication
- Title:
- National Toxicology Program, NTP Technical report on the toxicology and carcinogenesis studies of p-Nitrotoluene in F344/N rats and B6C3F1 mice.
- Author:
- US Department of Health and Human Services
- Year:
- 2 001
- Bibliographic source:
- NTP technical report No. 498, NIH Publication No. 01-4432.
- Reference Type:
- secondary source
- Title:
- European Union Risk Assessment Report - 2-Nitrotoluene
- Author:
- European Commission - European Chemicals Bureau
- Year:
- 2 008
- Bibliographic source:
- Office for Official Publications of the European Communities
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- Only 4 strain tested instead of 5. Positive control: 2-aminoanthracene was only indicator of efficiency of S9-mix. Individual plate reading not reported.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- 2-nitrotoluene
- EC Number:
- 201-853-3
- EC Name:
- 2-nitrotoluene
- Cas Number:
- 88-72-2
- Molecular formula:
- C7H7NO2
- IUPAC Name:
- 1-methyl-2-nitrobenzene
- Details on test material:
- - Name of test material (as cited in study report): o-nitrotoluene
- Analytical purity: > 99%
- Lot Number: A8A
Constituent 1
Method
- Target gene:
- his
Species / strain
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- microsomal enzyme reaction mix (S9-mix)
- Test concentrations with justification for top dose:
- Test concentrations:
SRI (LABORATORY): 0; 3.0; 10.0; 33.0; 100.0; 333.0; 666.0 µg/plate
EGG (LABORATORY): 0; 3.3; 10.0; 33.0; 100.0; 333.0µg/plate - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (2-AA): in all strains in presence of rat and hamster S-9. 4-Nitro-o-phenylenediamine (NOPD): on TA98, without S-9. sodium azide (SA) : on TA100 and TA 1535, without S-9. 9-aminoacridine (AAD) was tested on 1537, without S-9.
- Remarks:
- The actual concentration for each positive control chemical used for each strain and activation condition was selected by the individual laboratory based on dose-response curves generated at the beginning of the testing program (see table 1)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h
- Concentration of S9 mix: 10% for both Hamster S9 mix and Rat S9 mix
SELECTION AGENT (mutation assays): L- histidine
NUMBER OF REPLICATIONS: 2 trial per strain and 3 dishes per dose
DETERMINATION OF CYTOTOXICITY
- Method: other: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn - Evaluation criteria:
- A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.
An equivocal response was defined as an increase in revertants which was not dose-related, not reproducible, or was of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies is observed following chemical treatment.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: to select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.
Any other information on results incl. tables
Table 1.Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean±SEM) to o-nitrotoluene.
Abbreviations: NA, not activated; RLI, rat liver S-9, Aroclor1254induced; HLI, hamster liver S-9,Aroclor1254 induced, t=complete clearing background
| TA 100 | TA 1535 | TA 1537 | TA 98 | ||||||||
Dose | NA | RLI | HLI | NA | RLI | HLI | NA | RLI | HLI | NA | RLI | HLI |
0.0 | 138±16.3 | 123±7.9 | 134±6.2 | 25±3.8 | 10±0.6 | 14±1.7 | 8±1.5 | 10±1.5 | 13±1.2 | 14±2.7 | 25±0.7 | 28±1.2 |
3.3 | 135±4.3 | 149±7.8 | 127±2.3 | 26±2.5 | 10±1.2 | 15±3.2 | 8±2.1 | 8±0.9 | 9±2.1 | 20±3.8 | 29±1.2 | 31±1.9 |
10.0 | 139±5.8 | 128±3.2 | 118±7.6 | 23±3.3 | 9±1.7 | 17±2.5 | 5±1.8 | 6±1.2 | 12±0.0 | 16±1.9 | 27±0.3 | 31±7.1 |
33.3 | 122±6.9 | 147±21.7 | 134±2.5 | 24±1.7 | 10±3.3 | 20±1.5 | 6±1.2 | 7±2.1 | 9±0.6 | 22±1.2 | 27±5.2 | 32±1.9 |
100.0 | 121±11.1 | 142±5.2 | 135±9.3 | 22±4.9 | 19±2.0 | 16±1.5 | 8±1.3 | 6±0.9 | 8±1.2 | 20±1.2 | 25±1.9 | 34±2.3 |
333.3 | 132±9.2s | 115±11.0 | 148±3.3 | 23±2.8s | 12±1.2 | 16±2.5 | 6±0.3s | 7±0.9 | 11±0.9 | 12±1.8s | 27±3.7 | 36±3.5 |
POS | 2103±44.5 | 991±44.5 | 1900±92.3 | 1320±43.1 | 108±5.5 | 128±6.3 | 901±105.9 | 71±4.9 | 173±7.2 | 2119±51.4 | 874±29.2 | 1454±95.4 |
Table 2 Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean±SEM) to o-nitrotoluene.
Abbreviations: NA, not activated; RLI, rat liver S-9, Aroclor1254 induced; HLI, hamster liver S-9, Aroclor1254 induced, t=complete clearing background
| TA 100 | TA 1535 | TA 1537 | TA 98 | ||||||||
Dose | NA | RLI | HLI | NA | RLI | HLI | NA | RLI | HLI | NA | RLI | HLI |
0.0 | 120±4.7 | 117±7.8 | 126±9.1 | 15±1.7 | 10±1.9 | 12±2.1 | 5±2.6 | 6±0.9 | 8±2.6 | 22±2.7 | 24±2.6 | 28±4.6 |
3.3 | 118±8.5 |
|
| 10±1.5 |
|
| 4±0.6 |
|
| 24±1.9 |
|
|
10.0 | 105±4.7 | 130±3.8 | 133±4.7 | 11±1.2 | 10±2.8 | 6±1.7 | 3±0.7 | 5±1.3 | 9±2.3 | 16±0.9 |
29±2.3 | 35±1.7 |
33.3 | 110±7.5 | 133±7.5 | 125±0.9 | 14±1.5 | 10±2.1 | 10±2.6 | 4±0.3 | 6±0.7 | 6±1.2 | 17±1.5 |
30±0.9 | 29±1.8 |
100.0 | 104±11.2 | 147±5.3 | 125±13.9 | 18±5.2 | 7±2.9 | 10±1.5 | 3±0.9 | 8±0.7 | 8±2.6 | 36±22.3 |
34±2.7 | 27±1.3 |
333.3 | t | 114±1.9 | 125±7.0 | 0±0 | 11±1.5 | 8±0.7 | t | 6±1.0 | 6±1.5 | 0±0.0s |
31±3.8 | 28±2.6 |
666.0 |
| 119±2.5 | 137±5.7 |
| 9±3.0 | t |
| 6±1.3 | t |
|
31±0.5 | 0±0.0s |
POS | 424±16.2 | 900±15.3 | 1895±83.7 | 396±2.3 | 313±77.0 | 507±35.4 | 100±18.2 | 283±14.2 | 353±32.0 | 760±8.0 | 640±8.7 | 1761±147.7 |
Applicant's summary and conclusion
- Executive summary:
Haworth (1983):
2-nitrotoluene was tested in the Ames test similar to OECD guideline 471 with deviations (Only 4 bacterial strains were used. The highest dose tested was 10 mg/plate. 2-Aminoanthracene was tested as sole indicator of the efficacy of the S9-mix. 4-Nitro-o-phenylenediamine was tested on TA 98), using preincubation, in strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimirium.
Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, i.p., with Aroclor 1254. The concentrations of 2-nitrotoluene used were 0.0, 3.3, 10.0, 33.0, 100.0 and 333.0µg/plate in one experiment (EGG laboratory) and 0.0, 3.3, 10.0, 33.0, 100.0, 333.0, and 666.0 µg/plate (SRI laboratory).
The results were negative, 333.0µg/plate resulted toxic for the strains TA 100, TA 1537 and TA 98 without S9 mix.
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