N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jan to 21 Apr 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

258-110-1

EC Name:

N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

52697-38-8

Molecular formula:

C18H19BrN6O5

IUPAC Name:

N-{2-[(2-bromo-4,6-dinitrophenyl)diazenyl]-5-(diethylamino)phenyl}acetamide

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

According to the results of the pre-experiment the concentrations applied in the main experiments were chosen:
33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in DMSO (purity > 99 %, MERCK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
The test article precipitated at the higher concentrations in the overlay agar. The nondissolved particles of the test article had no influence on the data recording.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, TA 1535 and TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-NOPD

Remarks:

without S9, TA 1537 and TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level

DETERMINATION OF CYTOTOXICITY: Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

ACCEPTED CONDITIONS FOR EVALUATION:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Rationale for test conditions:

N.A.

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered nonmutagenic in this system.

A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice in strains TA 1535 and TA 1537).
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test article precipitated at the higher concentrations in the overlay agar. The nondissolved particles of the test article had no influence on the data recording.

Toxic effects, evident as a reduction in the number of revenants, were observed with and without S9 mix in strains TA 1535 and TA 1537 at 2500 and 5000 µg/plate, and in strain TA 100 with S9 mix in experiment II.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

 In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (60% purity) at concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test item was tested up to the limit concentration (5000 µg/plate).

Toxic effects, evident as a reduction in the number of revenants, were observed with and without S9 mix in strains TA 1535 and TA 1537 at 2500 and 5000 µg/plate, and in strain TA 100 with S9 mix in experiment II.

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

In both experiments, a substantial and dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 1535, TA 1537, TA 98, and TA 100.

However, overlapping toxic effects reduced the number of revertant colonies in strains TA 1535, TA 1537 (with and without metabolic activation), and TA 100 from 1000 up to 5000 µg/plate.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100.

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.