N,N'-1,4-phenylenebis[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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to recent OECD TG.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-1,4-phenylenebis[3-oxobutyramide]

EC Number:

246-438-8

EC Name:

N,N'-1,4-phenylenebis[3-oxobutyramide]

Cas Number:

24731-73-5

Molecular formula:

C14H16N2O4

IUPAC Name:

3-oxo-N-[4-(3-oxobutanamido)phenyl]butanamide

Details on test material:

- Name of test material (as cited in study report): Diacetessig-p-phenylendiamid (DAEP)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

On the day of the experiment, the test item Diacetessig-p-phenylendiamid (DAEP) was dissolved in DMSO (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. No precipitation of the test item was observed in the overlay agar

Controls

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, 2-aminoanthracene

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:

No statistical evaluation of the data is required.

Results and discussion

Test results

Test results 1

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Diacetessig-p-phenylendiamid (DAEP) 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.

In experiment II, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 100 in the solvent control with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

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 used.
Therefore, Di acetessig-p-phenylendiamid (DAEP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Di acetessig-p-phenylendiamid (DAEP) was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubationtest (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were 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, 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 Di acetessig-p-phenylendiamid (DAEP) 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 experiment II, the number of colonies did not quite reach the lower limit of our historical control data in strain TA 100 in the solvent control with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.