2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

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

Study period:

From 24 AUG 2012 to 13 SEP 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 471) and according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

EC Number:

229-419-9

EC Name:

2-[(4-methoxy-2-nitrophenyl)azo]-N-(2-methoxyphenyl)-3-oxobutyramide

Cas Number:

6528-34-3

Molecular formula:

C18H18N4O6

IUPAC Name:

2-[(4-methoxy-2-nitrophenyl)diazenyl]-N-(2-methoxyphenyl)-3-oxobutanamide

Test material form:

solid: bulk

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 and non-induced hamster liver S9

Test concentrations with justification for top dose:

Experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 4-Nitro-o-phenylene-diamine, methyl methane sulfonate

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: rat liver S9 mix: 2-aminoanthracene, hamster liver S9 mix: 2-aminoanthracene, congo red

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, plate incorporation, preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

8.4.1 Rat Liver S9 (Preparation by Harlan CCR)

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/mL (lot no. R 260412).

8.4.2 Hamster Liver S9 (Preparation by Harlan CCR)
The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters.
After decapitation of the anaesthetised animals the livers of the animals was removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionised water and homogenised. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9,000 g for 25 minutes at 4 °C. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.
The protein concentration in the S9 preparation was 24.0 mg/mL (lot no. H 020712).

8.4.3 Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

8.4.4 Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. and Prival and Mitchell .

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.

Executive summary:

This study was performed to investigate the potential of a test substance to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 and theEscherichia colistrain WP2uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I:           3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.
Experiment II:          10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.