2,6-dichloro-N-phenylaniline

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:

other information

Study period:

August 17 - October 16, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study compliant with TG guidelines and conducted under GLP in recognized industrial research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of 1983

Deviations:

yes

Remarks:

Statistical analysis was not performed, as it was considered to be inappropriate for this particular test system.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

,only EU Method B.14 of 1984

Deviations:

yes

Remarks:

Statistical analysis was not performed, as it was considered to be inappropriate for this particular test system.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

of 1987 (corrected at 52 FR 26150)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-dichloro-N-phenylaniline

EC Number:

239-349-0

EC Name:

2,6-dichloro-N-phenylaniline

Cas Number:

15307-93-4

Molecular formula:

C12H9Cl2N

IUPAC Name:

2,6-dichloro-N-phenylaniline

Details on test material:

- Name of test material (as cited in study report): PBS 3247.2
- Expiry date of the lot/batch: July 15, 1993
- Stability: On file with Sponsor (at that time i.e. CIBA-GEIGY Limited, Basle, Switzerland, Pharmaceuticals Division)
- Storage conditions: dry, room temperature

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine-requiring (auxotrophic) strains from Prof. B. Ames, Berkeley, CA., USA

Metabolic activation:

with and without

Metabolic activation system:

liver S9 post mitochondrial supernatant from male RAI rats (Tif: RAIf[SPF]) treated by intraperitoneal injection with Aroclor 1254 (500 mg/kg), 5 days prior to sacrifice for enzyme induction. The animals weighed 150 to 250 g.

Test concentrations with justification for top dose:

Pre-Experiment [Toxicity Test, without and with metabolic activation (S9)]:
TA100: 20.6, 61.7, 185, 556, 1667, 5000 ug/plate.
Experiments 1 and 2 [Mutagenicity Test, each experiment without and with metabolic activation (S9)]:
TA 98: 78.1, 156, 313, 625, 1250 µg/plate
TA 100: 19.5, 39.1, 78.1, 156, 313 µg/plate.
TA 1535: 78.1, 156, 313, 625, 1250 µg/plate
TA 1537: 9.77, 19.5, 39.1, 78.1, 156 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535, each without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9(5)-aminoacridine

Remarks:

TA 1537 without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 98, TA 100, TA 1537, each with metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 1535 with metabolic activation

Details on test system and experimental conditions:

It is probable that plate-incorporation tests were performed in the Pre-Experiment and both Main Experiments (1 and 2), as pre-incubation was not mentioned in the study report.

In a preliminary solubilisation test, a maximum solubility of test material in the vehicle, Dimethylsulfoxide (DMSO), of 50.0 mg/ml was attained. No precipitates or aggregates were noted at this or lower concentrations of test material in DMSO.

In the pre-experiment (Toxicity Test with TA 100), precipitation of the test substance was evident at 5000 µg/plate without and with metabolic activation (S9), but not at lower concentrations. No precipitation was observed in any tester strain used in the two Main Experiments (without and with metabolic activation).

Evaluation criteria:

Degree of increase in the number of revertant colonies in any tester strain. Dose dependency of any effects. Confirmation in independent repeat experiment.

Assay Acceptance Criteria:
A test was considered acceptable if the mean colony counts of the control values of all strains were within the acceptable ranges and if the results of the positive controls met the criteria for a positive response. In either case the final decision was based on the scientific judgement of the study Director. Acceptable ranges for negative controls were specified in the study report for each strain without and with metabolic activation.

Criteria for a Positive Response:
The test material was considered to be mutagenic in this test system if one or both of the following conditions were met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for
S. typhimurium TA 98, TA 1535 and/or TA 1537.
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control
at least by a factor of 1.5 for strain S. typhimurium TA 100.

Statistics:

Statistical analysis was not performed, as it was considered to be inappropriate for this test system.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >/= 61.7 µg/plate, starting concentrations varying between strain, metabolic activation and/or replicate experiment

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation (S9)

In the present study, the test material was tested for mutagenicity, both at non-toxic and cytotoxic test substance concentrations, in a reverse mutation assay using four different strains of S. typhimurium in the absence and presence of metabolic activation with S9. Mutagenicity of the test substance was not evident, as it did not induce any relevant increase in the number of revertant colonies in the four bacteria strains tested. Hence, there was no evidence of point mutations by base pair changes or frameshifts attributable to treatment with the test substance. Validity of the test procedures and conditions adopted and of the bacteria strains used was confirmed.

Executive summary:

The test material was tested for mutagenicity, both at non-toxic and cytotoxic test material concentrations (up to 1250 µg/plate) in an Ames test according to EU Method B.14 of 1984 and the corresponding OECD (471) and EPA-OTS Technical Guidelines. Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537) with and without metabolic activation with S9 was utilised.

 

The study was conducted in compliance with GLP. Reliability grade 1 was assigned to it.

 

Following a pre-experiment, two independent main experiments (Experiments 1 and 2; both were plate incorporation tests), were performed. Reliability grade 1 was assigned to the study.

 

Precipitation of the test material was not evident in the two main experiments in this study. Mutagenicity of the test material was not evident in this study, as it did not induce any relevant increase in the number of revertant colonies in the four bacteria strains tested. Hence, there was no evidence of point mutations by base pair changes or frameshifts attributable to treatment with the test material.

 

In the vehicle control plates receiving dimethylsulfoxide (DMSO) without the test material, the growth rates and mean numbers of revertant colonies were within expected ranges. In addition, the positive control mutagens induced distinctly higher numbers of revertant colonies than the vehicle controls, and the various sterility checks, sensitivity and resistance tests etc. adopted for quality check of the test system all produced results within the testing laboratory’s established limits. Therefore, validity of the conditions and procedures adopted in the present study and of the bacterial cultures used was confirmed.