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EC number: 202-762-1 | CAS number: 99-52-5
- 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
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- 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
Bacterial gene mutation assay was performed to determine the mutagenic nature of p-Nitro-o-toludine. The assay was performed using Salmonella typhimurium strain TA98 with and without S9 metabolic activation system. Suspension assay was performed with the test chemical dissolved in DMSO at dose level of 0 or 1 µg/plate. The plates were observed for a dose dependent increase in the number of revertants/plate. p-Nitro-o-toludine did not induce gene mutation in Salmonella typhimurium strain TA98 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from peer reviewed publication
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- Bacterial gene mutation assay was performed to determine the mutagenic nature of p-Nitro-o-toludine
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Name of test material: p-Nitro-o-toludine
- IUPAC name: 2-methyl-4-nitroaniline
- Molecular formula: C7H8N2O2
- Molecular weight: 152.152 g/mol
- Substance type: Organic
- Purity: No data available
- Impurities (identity and concentrations): No data available - Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 98
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- No data
- Metabolic activation:
- with and without
- Metabolic activation system:
- The postmitochondrial fraction (S9) was prepared from the liver of male Sprague-Dawley rats induced with PCB
- Test concentrations with justification for top dose:
- 0 or 1 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- 4-nitroquinoline-N-oxide
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): No data
DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: Triplicate
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data
NUMBER OF CELLS EVALUATED: No data
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- OTHER: No data - Rationale for test conditions:
- No data
- Evaluation criteria:
- The plates were observed for a dose dependent increase in the number of revertants/plate
- Statistics:
- No data
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- No data
- Conclusions:
- p-Nitro-o-toludine did not induce gene mutation in Salmonella typhimurium strain TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.
- Executive summary:
Bacterial gene mutation assay was performed to determine the mutagenic nature of p-Nitro-o-toludine. The assay was performed using Salmonella typhimurium strain TA98 with and without S9 metabolic activation system. Suspension assay was performed with the test chemical dissolved in DMSO at dose level of 0 or 1 µg/plate. The plates were observed for a dose dependent increase in the number of revertants/plate. p-Nitro-o-toludine did not induce gene mutation in Salmonella typhimurium strain TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.
Reference
Table: Mutagenicity of p-Nitro-o-toluidine in S. typhimurium strain TA98
Sample |
Dose (µg/plate |
His+revertants/plate |
|
Without |
With |
||
p-Nitro-o-toluidine |
1 |
28 |
39 |
DMSO |
|
14 |
28 |
4NQO |
0.5 |
111 |
|
AAF |
5 |
|
540 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in vitro:
Variuos peer reviewed publications were reviewed to determine the mutagenic nature of . The studies are as mentioned below:
Watanabe et al (Mutation Research, 1989) performed bacterial gene mutation assay to determine the mutagenic nature of p-Nitro-o-toludine (CAS no 99 -52 -9). The assay was performed using Salmonella typhimurium strain TA98 with and without S9 metabolic activation system. Suspension assay was performed with the test chemical dissolved in DMSO at dose level of 0 or 1 µg/plate. The plates were observed for a dose dependent increase in the number of revertants/plate. p-Nitro-o-toludine did not induce gene mutation in Salmonella typhimurium strain TA98 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
In another study by Yoshimi et al (Mutation Research, 1988), The hepatocyte/DNA repair test which measures unscheduled DNA synthesis (UDS) was performed to determine the genotoxicity of 2-methyl-4-nitroaniline using male ACI rat hepatocytes. The test was performed basically in accordance with the method of Williams et al. The test material was dissolved in DMSO and the positive control used was N-2-fluorenylacetamide. The isolated hepatocytes were allowed to attach for 2 h on plastic coverslips in primary culture using Williams' Medium E. The cultures were then washed and exposed to the test chemical and [Me- 3H]thymidine (10 µCi/ml; 49 Ci/mmole) for 20 h. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment. The data were expressed as the average net counts/nucleus for 3 coverslips + the standard deviation (50 cells/coverslip). The test chemical was considered positive when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controlsThe given test material 2-methyl-4-nitroaniline elicited negative DNA repair response in the rat hepatocyte/DNA repair test.
Matsushima et al (Mutagenesis, 1999) gave in vitro micronucleus test to determine the mutagenic nature of 2-methyl-4-nitroaniline. The test chemical dissolved in DMSO was exposed to the cells at a dose level of 250-1000 µg/mL( dose range represented for 6+18 h without S9 mix treatment protocol only) . Concurrent solvent and negative controls were included in the study. 1X 104– 1X 105cells were seeded in 60 mm plastic plates and treated with the test chemical on the second day. The cells were treated for 24 or 48 hrs without S9 and for 6 hrs with or without S9 mix followed by 18 hrs recovery period. The cells were then detached by trypsinization and treated with KCl hypotonic solution for 10 mins at room temperature. The hypotonized cells were fixed by atleast three changes of 1:3 acetic acid: ethanol. The cells were then suspended in methanol containing 1-2% acetic acid and air dried on clean glass slide. After overnight drying, the cells were stained with either acridine orange or Giemsa. All slides were coded and analyzed blind microscopically.The MNs were categorized in three groups: very small pinpoint inclusions stained homogeneously, typical i.e. smaller in diameter than ¼ of the normal main nucleus (type 2), large i.e. between ¼ and ½ the diameter of the normal main nucleus (type 3). The test compound induced polyploid cells with the 24 and 48 hrs continuous treatment: at 24 hrs a positive response (10.5%) was seen at 100 µg/mL and 48 hrs, a dose dependent response was induced (14.0% and 35.6% at 100 and 200 µg/mL respectively). No data was available at 48 hrs continuous treatment at over 100 µg/mL because of cytotoxicity. Based on the observations made, 2-methyl-4-nitroaniline did not induce micronuclei formation in Chinese hamster lung cell line (CHL/IU) and hence is considered to be negative for gene mutation in vitro.
In a study by Shibai-Ogata (Mutagenesis, 2011), In vitro Micronucleus test was performed to determine the mutagenic nature of 2-methyl-4-nitroaniline. The study was performed using maintained CHU/IU cells. The cells were exposed to the test chemical dissolved in DMSO at dose levels of 0, 78, 156, 313, 625, 1250, 2500, 5000 or 10000 µM. Cells were continuously treated with test chemical by employing four treatment procedures: brief treatment withor without S9 mix and two prolonged (24 or 48 h) treatments. Three thousand cells for 48-h treatment or 5000 cells for procedures other than the 48-h treatment were each seeded with 100 ll of medium in each well of 96-well microplates. Clear bottom black 96-well microplates were used for detection of MN and clear bottom white 96-well microplates were used for cytotoxicity. All incubations were carried out in a 5% CO2 humidified atmosphere at 37C. After the prescribed incubation time, the cells were washed with PBS once, fixed with 100% ethanol for atleast 30 min, followed by replacement of PBS and stored at 4C until the staining procedures.Fixed cells were washed and double stained with 5 µM Hoechst 33342 and 1 µM HCS CellMask Red in 100 µl PBS for 30 min. Hoechst 33342 was used for staining of nuclei and MN and HCS CellMask Red for staining of the cytoplasm. Then, the stain solution was replaced with 100 µl PBS to acquire fluorescence images. Fluorescence images of cells stained with Hoechst 33342 and CellMask Red were acquired with an IN Cell Analyzer 1000 using an X20 objective lens. The stored images were analysed employing IN Cell Developer Toolbox software. For automatic counting, eighteen fields per well (54 fields per concentration) were acquired and analysed and for visual observatoin, 500 cells were scored. Cytotoxicity was assessed as the reduction of cell viability. For evaluation of cell viability, microplate was treated with chemical in the same manner as the microplates used for acquiring fluorescence images. Following test chemical treatments, the microplates were washed with PBS once and 50 µl of PBS and 50 µl of CellTiter-Glo reagent were then added followed by plate shaking for 10 min, and the amount of intracellular ATP in surviving cells per well was measured. Intracellular ATP was estimated quantitatively using a CellTiter-Glo Luminescent CellViability Assay kit and luminescence was measured using Varioskan Flash Microplate Multimode Readers. Relative survival was calculated as the ratio of the amount of luminescence, which depends on the amount of intracellular ATP, in treated cells versus the amount of luminescence in solvent control cells, expressed as a percentage. An independent assay was considered to be negative when the assay acceptance criteria were satisfied and there was no significant increase in the frequency of MN cells as compared to controls. 2-methyl-4-nitroaniline did not induce an increase in the frequency of MN cells as compared to controls during the brief treatment with and without S9 activation and also during the 48 hrs treatment without S9 mix. It however showed an ambiguous response during 24 hrs treatment without S9 mix.
Matsushima et al (Mutagenesis, 1999) also gave chromosome aberration test to determine the mutagenic nature of 2-methyl-4-nitroaniline. The test chemical dissolved in DMSO was exposed to the cells. Concurrent solvent and negative controls were included in the study. 1X 104– 1X 105cells were seeded in 60 mm plastic plates and treated with the test chemical on the second day. The cells were treated for 24 or 48 hrs without S9 and for 6 hrs with or without S9 mix followed by 18 hrs recovery period. The cells were then detached by trypsinization and treated with KCl hypotonic solution for 10 mins at room temperature. The hypotonized cells were fixed by atleast three changes of 1:3 acetic acid: ethanol. The cells were then suspended in methanol containing 1-2% acetic acid and air dried on clean glass slide. After overnight drying, the cells were stained with either acridine orange or Giemsa. All slides were coded and analyzed blind microscopically. Short treatment of the test chemical with S9 mix yielded marginal responses for structural and numerical aberrations, with S9 the response was negative. The test chemical induced polyploidy cells at 100µg/mL with 24 hrs (11%) and 48 hrs (10.3%) continuous treatment.
In an abstract mentioned in European Environmental Mutagen Society (EEMS) publication (1989) for the target chemical, Gene mutation toxicity study was performed to determine the mutagenic nature of 4-nitro-o-toluidine (4-NT) using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 activation system. The test was performed at dose levels of 0.25 – 2.5 mM. The mutagenicity of 4-NT was higher with strain TA 98 where 2.5 mM 4-NT induced without S9-mix a 10-fold increase of revertant numbers as compared to the control value. In the presence of S9-mix, 4-NT was less mutagenic. No mutagenic activity was noted in strain TA100 in the absence of S9 metabolic activation system.
In the above mentioned publication (European Environmental Mutagen Society (EEMS) publication (1989)), Chromosome aberration study was performed to determine the mutagenic nature of 4-nitro-o-toluidine (4-NT) using human lymphocytes. The study was performed at dose levels of 0.25 – 4 mM in the presence and absence of S9 activation system. 4-nitro-o-toluidine elicited chromosomal aberrations in human lymphocytes with and without S9 activation system.
Based on the data summarized for the target chemical, 2-methyl-4-nitroaniline does not exhibit gene mutation in vitro. Though some positive effects have been mentioned for the target chemical in the data mentioned in European Environmental Mutagen Society (EEMS) publication but the information available to predict the positive nature is very less and unclear. Hence based on majority of data summarized, the target chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.
Justification for classification or non-classification
Based on the data summarized for the target chemical, 2-methyl-4-nitroaniline does not exhibit gene mutation in vitro. Though some positive effects have been mentioned for the target chemical in the data mentioned in European Environmental Mutagen Society (EEMS) publication but the information available to predict the positive nature is very less and unclear. Hence based on majority of data summarized, the target chemical is not likely to classify as a gene mutant in vitro as per the criteria mentioned in CLP regulation.
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