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EC number: 202-808-0 | CAS number: 99-99-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Reverse gene mutation assay; OECD 471: Negative (Key: Haworth, 1983, rel.2)
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline with acceptable restrictions (no data on substance purity, cells harvested after only 1 mitotic cycle, only 100 metaphases per concentration was evaluated)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- (cells harvested after only 1 mitotic cycle, only 100 metaphases per concentration was evaluated)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- (CHO-W-B)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 was from the livers of Aroclor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Without S9 first and second trial: 300; 400; 500 µg/ml;
Without S9 second trial: 300, 400, 500 µg/ml
With S9 second trial: 400, 500, 550 µg/ml - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Migrated to IUCLID6: -S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Migrated to IUCLID6: +S9
- Details on test system and experimental conditions:
- Each concentration was test in duplicate cultures.
Doses were based on observations of cell confluence and mitotic cell availabiity in the SCE test (performed alongside chromosome aberration test). The doses for the aberration tests were usually spaced more closely than the half-log series of the preliminary SCE test and extended into the toxic range. In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test).
The cell harvest time for the aberration test was 10.5 h after the beginning of treatment.
This yielded cells in their first mitosis. In the presence of S9, cells were incubated with onitrotoluene or solvent for 2 hours at 37° C. Cells were then washed, medium was added, and incubation was continued for 8.5 hours. Colcemid was added for the last 2 to 3 hours of incubation before harvest. In the absence of S9, cells were incubated with o-nitrotoluene or solvent for 8.5 hours at 37° C. Cells were then washed and fresh medium containing
Colcemid was added for an additional 2 hours followed by harvest. Cells were collected by mitotic shake-off.
Slides were stained with Giemsa (6%), and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (mitomycin or cyclophosphamide) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), "other" (includes pulverized chromosomes), and "total." Gaps and endoreduplications were recorded but were not included in the totals. Aberrations in polyploid cell were not scored but used metaphases with 19-23 chromosomes (the modal number being 21) - Evaluation criteria:
- Chromosomal aberration data is presented as percentage of cells with aberrations. Both the dose-response curve and individual dose points were statistically analyzed. For a single trial, a statistically significant (P<05) difference for 1 dose point and a significant trend (P<0.015) was considered weak evidence for a positive response (W+); significant differences for two or more doses indicated the trial was positive (+)
- Statistics:
- For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations versus the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al [1983, pp 714-715].
The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. For data analysis, the "total" aberration category, and the criterion for a positive response was that the adjusted P value be <= 0.05. - Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- weak positive in the first trial and positive in the second trial
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Executive summary:
Effects on chromosomes were studied in Chinese hamster ovary (CHO) cells in tests corresponding to the current guideline (Galloway et al., 1983; 1987; US Department of Health and Human Services, 1992; 2002). In the presence, but not in the absence of metabolic activation, 4-nitrotoluene induced chromosome damage at cytotoxic dose levels.
- Endpoint:
- in vitro DNA damage and/or repair study
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline with acceptable restrictions (no data on substance purity, no data on cytotoxicity)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
- Deviations:
- yes
- Remarks:
- (no data on cytotoxicity)
- Principles of method if other than guideline:
- Testing was performed as reported by Loveday et al. (1989) Environ. Molec. Mutagen. 13, 60-94
- GLP compliance:
- yes
- Type of assay:
- sister chromatid exchange assay in mammalian cells
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- (CHO-W-B)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 was from the livers of Aroclor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Without S9 first trial : 50; 167; 500 µg/ml;
without S9 second trial: 200, 300, 400, 500 µg/ml;
With S9 first trial: 50; 165; 500 µg/ml;
with S9 second trial: 600, 700 µg/ml;
with S9 third trial: 550, 600, 650 µg/ml - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Migrated to IUCLID6: -S9
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- Migrated to IUCLID6: +S9
- Details on test system and experimental conditions:
- Each test point was performed in duplicates
In the absence of S9, cells were incubated with o-nitrotoluene or solvent for 2 hours at 37° C. Then 5-Bromodeoxyuridine BrdU was added and incubation was continued for another 23.5 hours making a total incubation time of 25.5h. Cells were washed, fresh medium containing BrdU and Colcemid was added, and incubation was continued for 2 to 3 hours.
In the presence of S9, cells were incubated with o-nitrotoluene or solvent for 2 hours at 37° C. The cells were then washed, and medium containing BrdU was added. Cells were incubated for a further 25.5 hours, with Colcemid present for the final 2 to 3 hours.
Immediately before the cells were harvested, the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could be made from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCI), cells were fixed in 3:1 methanol : glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with "dilute" Hoeschst 33258 (0.5 µg/ml in Sorensen's buffer, pH 6.8) and examined by fluorescence microscopy to assess ceII cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25- 26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis (34 h) - Evaluation criteria:
- For individual doses, absolute increases in SCEs per chromosome of 20% or more over the
solvent control were considered significant. - Statistics:
- For SCE data, a linear regression trend has been used (trend test) of SCEs per chromosome vs log of the dose [Armitage, 1955. Biometrics 11:374-386]
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- weak positive in the first trial and positive in the second trial
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- cell cycle delay
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- other: Second trial. weak positive [Significant increase of SCE rate of SCE/chromosome over controls in the presence of metabolic activation at highest concentration]
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- cell cycle delay
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- third trial [Significant increase of SCE rate of SCE/chromosome over controls in the presence of metabolic activation)]
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- first trial
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Executive summary:
4-Nitrotoluene induced increases in sister chromatid exchange rates in Chinese Hamster Ovary (CHO) cells with and without S9-mix at doses that induced cell cycle delay, which is an indication of cytotoxicity (Galloway et al., 1987)
- Endpoint:
- in vitro gene mutation study in mammalian cells
- 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 restrictions (colony size not reported)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- yes
- Remarks:
- Colony size not reported
- Principles of method if other than guideline:
- Protocol presented by Myhr et al., Prog. Mutat. Res. 5, 555-568 (1985), highest dose was determined by limit of solubility and toxicity, colony size not reported
- GLP compliance:
- not specified
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase (TK)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9-fraction was routinely prepared from male Sprague-Dawley rats and male Syrian hamster that were injected , i.p., with Aroclor 1254.
- Test concentrations with justification for top dose:
- -S9-mix: ethanol: (1) 0, 75, 100, 150, 180, 200, 240 (2) 0, 25, 50, 75, 100,
150, 250 ug/ml
+S9-mix: I. acetone: (1)(2)(3) 0, 50, 75, 100, 150, 200, 300, 500,
II. Ethanol: 50, 100, 150, 200, 250, 300 ug/ml - Vehicle / solvent:
- acetone; ethanol
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: in the presence of S9-mix: methylcholantrene in the absence of S9-mix: methyl methansulfonate
- Details on test system and experimental conditions:
- L5178Y mouse lymphoma cells were maintained at 37° C as suspension cultures in supplemented Fischer´s medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.
All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated.
Treated cultures contained 6 × 106 cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with p-nitrotoluene continued for 4 hours, at which time the medium plus p-nitrotoluene was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37° C in 5% CO2 for 10 to 12 days. The test was initially performed without S9. Because a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254-induced male F344 rats - Evaluation criteria:
- All data were evaluated statistically for trend and peak responses
- Statistics:
- Both responses had to be significant (P=0.05) for p-nitrotoluene to be considered positive, i.e., capable of inducing TFT resistance. A single significant response led to a call of ¿questionable,¿ and the absence of both a trend and peak response resulted in a ¿negative¿ call.
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Solvent acetone: trial 1,2,3: 300 ug/ml: pracipitate of 4-nitrotoluene; 500 ug/ml: lethal; 50-200 ug/ml: significant positive response
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Remarks:
- Solvent ethanol: Trial (1): 250, 300 ug/ml: precipitate of 4-nitrotoluene; 50, 100 ug/ml and 200, 250, 300 ug/ml: significant positive response,
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Solvent ethanol: Trial (1): 240 ug/ml: significant positive response
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- no cytotoxicity or precipitation observed
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Solvent ethanol: Trial (2): 150, 250 ug/ml: significant positive response
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- Solvent ethanol: 150 ug/ml
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Executive summary:
4-Nitrotoluene was tested positive in a L5178Y mouse lymphoma assay in the presence of rat liver S9 mix. As the colony size is not reported, it is unclear whether this response can be attributed to gene mutations or cytogenetic effects (U.S. Department of Health and Human Services, 1992; 2002).
- 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 guidelien study with acceptable deviations (only four strains)
- 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-aminoantracene was only indicator of efficiency of S9-mix. Individual plate reading not reported.
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his
- Species / strain / cell type:
- other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537
- 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:
- 0, 3.3, 10.0, 33.0, 100.0, 333.3, 500.0 ug/plate
- Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- 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. - 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 examined
- Positive controls validity:
- valid
- 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 examined
- Positive controls validity:
- valid
- 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 examined
- Positive controls validity:
- valid
- 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 examined
- 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.
- Executive summary:
Haworth (1983):
Ames test was performed with the Salmonella typhimuriumstrains TA1535, TA1537, TA98, and TA100 at 0, 3.3, 10, 33.3, 100, 333.3, and 500 µg 4 -nitrotoluene/plate with and without S-9 from Aroclor 1254 induced rat and hamster livers using the preincubation procedure. Test doses were chosen following checks for toxicity: in the absence of toxicity a maximum of 10 mg/plate was used. The result was negative.
The test was conducted according to OECD guideline 471 with deviations (Only 4 strains tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix. Individual-plate-readings were not reported.)
Referenceopen allclose all
strain/cell type: Chinese hamster Ovary (CHO)
strain/cell type: Chinese hamster Ovary (CHO)
strain/cell type: mouse lymphoma L5178Y cells
| TA 100 | TA 1535 | TA 1537 | TA 98 | ||||||||
Dose | NA | RLI | HLI | NA | RLI | HLI | NA | RLI | HLI | NA | RLI | HLI |
0.0 | 127±13.3 | 128±15.3 | 145±8.7 | 22±2.0 | 9±0.3 | 11±2.1 | 5±1.9 | 7±1.7 | 4±1.5 | 13±1.7 | 23±5.6 | 23±2.1 |
3.3 | 125±10.5 |
|
| 24±0.9 |
|
| 4±2.6 |
|
| 11±0.6 |
|
|
10.0 | 141±5.3 | 118±9.5 | 131±3.8 | 20±1.3 | 9±1.9 | 15±0.7 | 5±0.9 | 7±1.9 | 7±0.7 | 15±1.7 | 23±2.3 | 23±2.5 |
33.3 | 109±6.5 | 131±3.9 | 134±6.9 | 19±1.2 | 14±2.5 | 10±1.0 | 4±1.5 | 5±2.0 | 6±0.3 | 15±1.2 | 20±1.2 | 22±0.3 |
100.0 | 124±9.8 | 137±6.2 | 136±5.7 | 19±3.0 | 11±1.7 | 12±2.2 | 3±1.2 | 6±1.0 | 7±0.0 | 14±1.3 | 24±4.1 | 27±4.5 |
333.3 | 147±3.8s | 137±4.5 | 164±5.5 | 21±2.7s | 16±1.5 | 17±1.9 | 4±0.7 | 6±2.7 | 5±2.5 | 11±2.0 | 22±0.9 | 27±1.2 |
500.0 |
| 136±5.0s | 143±11.9s |
| 10±1.5s | 11±2.6s |
| 6±1.0s | 6±1.0s |
| 16±2.0s | 27±5.0s |
POS | 1343±30.9 | 1061±52.5 | 1191±27.7 | 980±33.6 | 82±3.5 | 49±2.5 | 379±61.2 | 77±6.1 | 133±3.8 | 1347±43.7 | 752±10.7 | 1045±12.6 |
Table 1 Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean ±SEM) to p-nitrotoluene
Abbreviations: NA, not activated; RLI, rat liver S-9, Aroclor1254 induced; HLI, hamster liver S-9, Aroclor1254 induced, t=complete clearing background
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro, 4-nitrotoluene showed no mutagenic effect in good quality Ames tests with Salmonella typhimurium and Escherichia coli, with and without metabolic activation. In cultured mammalian cells, 4-nitrotoluene has demonstrated the potential to cause mutagenicity in the presence of metabolic activation. The chemical did not induced unscheduled DNA synthesis in hepatocytes.
In vivo, 4-Nitrotoluene had no genotoxic activity. The substance did not induce micronuclei in rat and mice bone marrow cells in studies performed according to the current standard (OECD TG 474) and it did not induce unscheduled DNA synthesis in rat ex vivo hepatocytes.
Short description of key information: sourced from OECD SIDS
In vitro Studies
In tests performed according to current standards, 4-nitrotoluene was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA102, TA104 and in Escherichia coli
WP2uvra (dose-range, with and without metabolic activation: 0.0763 - 5000 µg/plate (JETOC 1996); 3.3 - 1000 µg/plate (U.S. Department of Health and Human Services, 2002). The studies gave no indication of gene mutation with and without metabolic activation. The positive controls were functional.
In earlier studies also some positive responses were obtained, mainly in Salmonella typhimurium
TA100. These results, however, were either not well documented, or were obtained with nonspecified test material or in non-standard test systems (e.g. Shimizu and Yano, 1986; Nohmi et al., 1984; Melnikov et al., 1981). These positive findings were therefore considered as of less relevance as compared to the data from tests performed in accordance with current standards. Overall, based on the studies of best quality, 4-nitrotoluene is considered as non-mutagenic in the Ames test system.
4-Nitrotoluene was tested positive in a L5178Y mouse lymphoma assay in the presence of rat liver
S9 mix. As the colony size is not reported, it is unclear whether this response can be attributed to gene mutations or cytogenetic effects (U.S. Department of Health and Human Services, 1992;
2002).
Effects on chromosomes were studied in Chinese hamster ovary (CHO) cells in tests corresponding to the current guideline (Galloway et al., 1983; 1987; US Department of Health and Human Services, 1992; 2002). In the presence, but not in the absence of metabolic activation, 4-nitrotoluene induced chromosome damage at cytotoxic dose levels.
4-Nitrotoluene induced increases in sister chromatid exchange rates in Chinese Hamster Ovary
(CHO) cells with and without S9-mix at doses that induced cell cycle delay, which is an indication of cytotoxicity (Galloway et al., 1987; US Department of Health and Services, 1992; 2002).
Indicator test: 4-nitrotoluene caused no unscheduled DNA synthesis (UDS) in cultured primary hepatocytes of rats (Doolittle et al., 1983).
Conclusion
In vitro, 4-nitrotoluene showed no mutagenic effect in good quality Ames tests with Salmonella typhimurium and Escherichia coli, with and without metabolic activation. In cultured mammalian cells, 4-nitrotoluene has demonstrated the potential to cause mutagenicity in the presence of metabolic activation. The chemical did not induced unscheduled DNA synthesis in hepatocytes.
In vivo Studies
There are bone marrow micronucleus tests available, that were performed with male rats and male mice according to OECD TG 474. 4-Nitrotoluene caused no increases in micronucleated polychromatic erythrocytes (PCEs) in the bone marrow of male rats given 0, 150, 300, or 600 mg/kg bw by intraperitoneal injection three times at 24 hour intervals. In male mice, treated with 0, 150, 300, or 600 mg/kg bw by intraperitoneal injection three times at 24 hour intervals, results of a first trial were considered positive, based on the responses of the two lowest doses; the trend test was not significant due to a downturn at the highest dose level. A second trial failed to induce a significant increase in micronucleated PCEs over the same dose range. Therefore the authors concluded the overall results as negative. Neither in the study with rats nor in the studies with mice were any signs of toxicity reported (U.S. Department of Health and Human Services, 2002). Indicator test 4-Nitrotoluene did not induce unscheduled DNA synthesis (UDS) in hepatocytes of rats after single oral treatment by gavage with 0, 100, 200, 500 mg/kg bw dissolved in corn oil or 0, 50, 200, 1000 mg/kg bw in corn oil (US Department of Health and Human Services, 1992; Mirsalis, 1989).
Conclusion
In vivo, 4-Nitrotoluene had no genotoxic activity. The substance did not induce micronuclei in rat and mice bone marrow cells in studies performed according to the current standard (OECD TG 474) and it did not induce unscheduled DNA synthesis in rat ex vivo hepatocytes.
Justification for classification or non-classification
Harmonised classification:
The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).
Self classification:
Based on the available information no additional self-classification is proposed according to the CLP and to the GHS.
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