2-[(2-methoxy-4-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:

29 November 2006 to 08 December 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetz and OECD Principles of Good Laboratory Practice

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

228-768-4

EC Name:

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

Cas Number:

6358-31-2

Molecular formula:

C18H18N4O6

IUPAC Name:

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

Test material form:

solid: nanoform, no surface treatment

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment and Experiment I: 0 (control), 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II: 0 (control), 33, 100, 333, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below for additional information

Details on test system and experimental conditions:

The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: pre-incubation test with and without non-induced hamster liver S9 mix

Hamster liver S9 mix, but not rat liver S9 mix, contained the reductive agent FMN.


DURATION
- Preincubation period: 30 min, 30°C
- Exposure duration: after solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more

POSITIVE CONTROL SUBSTANCES:
without metabolic activation: sodium azide (TA 1535, TA 100), 4-nitro-o-phenylene-diamine ((TA 1537, TA 98), methyl methane sulfonate (WP2 uvrA);
with metabolic activation: 2-aminoanthracene (all strains with rat liver S9 mix and TA 1535, TA 100, TA 1537, WP2 uvrA with hamster liver S9 mix), cKongo red (TA 98 with hamster liver S9 mix)

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I, TA 1537, without metabolic activation: minor reduction in number of revertants at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

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

Any other information on results incl. tables

Mean mutant number ratios treated/solvent control

Exp. I: plate incorporation method without S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.0 -- 0.8 -- 0.8 -- 1.1 -- 0.8 -- 0.7 -- 0.9 -- 0.7

TA1537 -- 1.5 -- 1.1 -- 0.9 -- 0.9 -- 0.9 -- 1.3 -- 0.9 -- 0.4

TA98 -- 0.9 -- 1.0 -- 1.0 -- 1.1 -- 0.8 -- 0.9 -- 0.9 -- 0.7

TA100 -- 0.8 -- 0.9 --1.0 -- 1.1 -- 0.9 -- 1.0 -- 0.9 -- 0.8

WP2uvrA -- 1.0 -- 1.0 -- 1.1 -- 1.2 -- 0.9 -- 0.9 -- 0.8 -- 0.8

Exp. I: plate incorporation method with rat S9 mix

Concentrations given in µg/plate

Strain -- 3 -- 10 -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.1 -- 1.1 -- 1.1 -- 1.0 -- 1.1 -- 1.2 -- 1.2 -- 0.7

TA1537 -- 1.1 -- 0.9 -- 1.1 -- 1.3 -- 1.1 -- 0.9 -- 0.9 -- 0.7

TA98 -- 1.1 -- 1.1 -- 1.1 -- 1.0 -- 0.9 -- 0.9 -- 1.0 -- 0.8

TA100 -- 1.0 -- 1.1 --1.0 -- 1.0 -- 1.0 -- 1.0 -- 0.9 -- 0.8

WP2uvrA -- 1.0 -- 1.0 -- 0.9 -- 0.9 -- 0.9 -- 0.8 -- 0.8 -- 0.8

Exp. II: pre-incubation method without S9 mix

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 1.2 -- 0.9 -- 1.0 -- 0.9 -- 1.0 -- 0.9

TA1537 -- 1.2 -- 1.3 -- 1.4 -- 1.4 -- 1.1 -- 0.9

TA98 -- 1.0 -- 1.2 -- 0.9 -- 1.1 -- 0.8 -- 0.7

TA100 --1.2 -- 1.1 -- 1.1 -- 1.1 -- 0.9 -- 1.0

WP2uvrA -- 1.2 -- 0.9 -- 1.1 -- 0.9 -- 1.0 -- 0.9

Exp. II: pre-incubation method with hamster S9 mix

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA1535 -- 0.9 -- 0.8 -- 0.7 -- 0.7 -- 0.7 -- 0.7

TA1537 -- 1.3 -- 1.0 -- 1.1 -- 0.8 -- 0.8 -- 0.8

TA98 -- 1.0 -- 1.0 -- 1.0 -- 1.0 -- 0.7 -- 0.7

TA100 --1.0 -- 0.8 -- 1.0 -- 0.9 -- 0.8 -- 0.7

WP2uvrA -- 1.2 -- 0.8 -- 0.7 -- 1.0 -- 0.9 -- 0.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by
frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the 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 the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

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:

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

Experiment II: 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 metabolic activation in both experiments.

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in all strains. Only in experiment I in strain TA 1537 in the absence of metabolic activation a minor reduction in the number of revertants, were observed at 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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 and showed a distinct increase of induced revertant colonies.