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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
Reference
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:
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

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

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.

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.