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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)

Data source

Reference Type:
Comparison of the mutagenic specificity induced by four nitro-group-containing aromatic amines in Salmonella typhimurium his genes
King-Thom Chung, Thomas J. Hughes , Larry D. Claxton
Bibliographic source:
Mutation Research, 465, 2000. 165–171

Materials and methods

Test guideline
according to
other: as mentioned below
Principles of method if other than guideline:
2NPD previously was shown to be direct-acting mutagens in S. typhimu rium strain TA100. In this study, the compoundwere further tested for mutagenicity on Xenometrix strains of S. typhimurium TA7001 and TA7003.
GLP compliance:
Type of assay:
bacterial gene mutation assay

Test material

Details on test material:
Name of test material (as cited in study report):2 nitro p phenylene diamine (2NPD)Substance type: OrganicPhysical state: SolidPurchased from Sigma Chemical Company


Target gene:
Plate incorporation
Species / strain
Species / strain / cell type:
other: Xenometrix strains of S. typhimurium TA7001 and TA7003
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
600, 900,1200,1500,1800µg/plate
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: 4NQO = 4-nitroquinoline-N-oxide; MMS = methylmethane sulfonate; Anth = anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper diskAgar plateNUMBER OF REPLICATIONS: triplicate platesOTHER EXAMINATIONS:This research uses these strains to examine the mutational specificity of the aromatic amines 2NPD,3NPD, 4NPD, and DNBA which had been shown to cause mis-sense mutations in Salmonella tester strain TA100.The predominant mutational types were CG – TA transitions and CG - AT transversions
Evaluation criteria:
Test compounds were judged to be positive when they showed a significant dose response trend and fulfilled other requirements for an adequate assay. The Xenometrix strains produce low spontaneous counts; however, the control means do vary from assay to assay. Because of these factors and because the authors do not yet have an extensive historical database for control values, an absolute increase of 10 colonies was required in addition to the significant dose response. Those responses, demonstrating a significant dose–response trend and not showing the required increase, were classified as borderline. Unless otherwise noted, the discussion will assume that the borderline results are equivalent to nonmutagenic results.
The mean ± standard deviation of the numbers of revertant colonies from a final definitive experiment

Results and discussion

Test results
Species / strain:
other: Xenometrix strains of S. typhimurium TA7001 and TA7003
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
No data
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'. Remarks: Bacteria tester strains

Applicant's summary and conclusion

Interpretation of results (migrated information):negative with and without2NPD was detected to be non mutagenic for the strains TA7001 and TA7003.
Executive summary:

2-nitro-p-phenylenediamine (2NPD) is a direct-acting mutagen in Salmonella typhimurium strain TA100. The compound was tested further using the Xenometrix strains of S. typhimurium: TA7001 and TA7003, with and without S9 mix in the plate incorporation assay. Each of the strains detects only one unique base substitution event and only that event.


The standard S. typhimurium plate incorporation assay was used in this study. Mixtures of cell suspension(100µl), sample solution(up to 100ml)the 0.1 M sodium phosphate buffer, and the Aroclor - induced rat liver S9_7%. _500ml.were added to the top agar and the mixtures were poured onto the bottom agar. All experiments were performed with triplicate plates at five doses. The slope of the dose–response curvethe number of revertants per microgram was calculated by least-squares linear regression from the first linear portion of the dose– response curve using the GeneTox Manager software. The data represent the mean±standard deviation of the numbers of revertant colonies from a final definitive experiment.


The compound was non mutagenic to TA7001 and TA7003.

As per the CLP classification, the test material is not likely to classify for gene mutation in vitro.