N-(4-aminophenyl)aniline

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:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

other: assay protocol well documented comparable to current guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-aminophenyl)aniline

EC Number:

202-951-9

EC Name:

N-(4-aminophenyl)aniline

Cas Number:

101-54-2

Molecular formula:

C12H12N2

IUPAC Name:

N1-phenylbenzene-1,4-diamine

Details on test material:

purity: 99.28%, lot/batch no.:51

Method

Target gene:

TA98, TA100, TA1535 and TA 1537

Species / strain

Species / strain / cell type:

other: TA98, TA100, TA1535 and TA 1537

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from aroclor 1254 induced livers

Test concentrations with justification for top dose:

0, 0.05, 0.15, 0.5, 1.5, 5 mg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide, 2-acetylaminofluorene, benzo(a)pyrene, sodium nitrite, 2-aminoanthracene, 9-aminoacridine

Details on test system and experimental conditions:

The Salmonella typhimurium test strains (TA98, TA1 00, TA1 535 and TA1 537) were obtained from the laboratory of Dr. B. N. Ames (Berkeley, CA). The cultures used were inoculated from frozen permanent stocks and grown in nutrient broth at 37°± 1°C in a
shaking incubator. The proper phenotype of each culture was verified by tests for crystal violet sensitivity, ampicillin resistance, requirement for histidine and biotin and spontaneous reversion frequency.

Plate Incorporation Tests
The general procedures used were basically those described by Ames et al. (Ref. 1). Plate incorporation tests were performed by mixing 0.1 ml of bacterial culture and, if appropriate, 0.5 ml of S-9 Mix (as described above) with 2 ml of histidine-biotin top
agar (0.5% (w/v) NaCI, 0.6% (w/v) Difco agar, 0.05 mM L-histidine-HCI, 0.05 mM ( biotin) maintained at 44-48°C. The mixture was poured onto minimal glucose agar plates (Vogel-Bonner medium E of Ref. 2 with 2% glucose and 1.5% Difco agar).
Initially, a toxicity test was conducted with test strain TA100 with and without S-9 Mix to define dose levels for mutagenicity testing. The toxicity screen used the same general r procedures as those described above. Single plates were prepared for each
L S-9/dose level combination for the toxicity test. Toxicity was judged qualitatively by visual examination of the background lawn and consideration of reduction in revertant colonies. Once dose levels were established using information from the toxicity
screen, mutagenicity testing was conducted using plate incorporation assay methods and four test strains (TA98, TA1 00, TA1 535 and TA1 537) both with and without S-9 Mix. In the mutagenicity test, three replicate plates were prepared for each
strain/S-9/test material dose level combination. Concurrent positive and negative controls (solvent and non-solvent) were conducted for all mutagenicity tests to demonstrate strain sensitivity and metabolic activation system capability. Plates were
examined after at least 48 hrs at 37°±1 °C. Solvent control plates used the same solvent and volume per plate as that selected for the test material. Two separate experiments were performed for each strain/S-9 combination to evaluate
reproducibility of results.

Statistics:

Statistical analysis was performed on plate incorporation assay results after transforming revertant/plate values as 10gb (revertants/plate). Analysis included
Bartlett’s test for homogeneity of variance (Ref. 3) and comparison of treatments with controls using within-levels pooled variance and a one-sided t-test (Ref. 4-6). Grubbs’
test was performed to determine if outliers were present (Ref. 7). Statistical significance of dose response was evaluated by regression analysis for logbO
transformed doses and revertants/plate (Ref. 8). A critical level of p0.01 was used in determining statistical significance. Results with
p0.05 are also indicated to assist in interpretation of results. As a general guide, Li results were considered clearly positive for a strain/microsome combination if
revertants/plate values were significantly elevated over control values (p0.01) at r three or more treatment levels, and there was a statistically significant dose response
L (pO.01). Other evaluation factors included consideration of the reproducibility of increases in revertants/plate, whether data are consistent with a biologically plausible
r r dose response, and the magnitude of the response. The criteria given above provide L guidance for evaluating the data. The study director made the final determination as to
whether a treatment-related mutagenic response was observed based on these general criteria as well as scientific evaluation of the data.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation: no precipitation of the test material was observed up to the maximum level tested 5 mg/plate

Toxicity: was observed at 1.5 mg/plate or higher without metabolic activation, and at 5 mg/plate with S9 mix.

No significant mutagenicity was observed in either in the inital or the subsequent confirmation assays for TA98, TA100, TA1535 and TA 1537 in the absence or presence of S9 mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

The test material, 4-ADPA, was tested in Ames/Salmonella plate incorporation assays using test strains TA98, TA100, TA1535 and TA1537 in the presence and absence of an Aroclor 1254-induced rat liver homogenate activation system (S-9 Mix). The maximum dose levels chosen for mutagenicity testing, 1.5 mg/plate without S-9 Mix and 5 mg/plate with S-9 Mix, gave clear indications of toxic responses for all strain/activation combinations. No significant mutagenicity was observed in either the initial or the subsequent confirmation assays for TA98, TA 100, TA1535 and TA1537 in the absence or presence of S-9 Mix. These results indicate that 4-ADPA is not a mutagen in the Ames/Salmonella plate incorporation assay under the experimental conditions.