N,N-Diethylacrylamide

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:

From 1993-07-06 to 1993-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-diethylprop-2-enamide

EC Number:

679-769-5

Cas Number:

2675-94-7

Molecular formula:

C7-H13-N-O

IUPAC Name:

N,N-diethylprop-2-enamide

Specific details on test material used for the study:

Batach No.: not specifed
Purity: not specifed

Method

Target gene:

Histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli (E. coli) strain

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:

other: S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver metabolic activation system (S-9 mix)

Test concentrations with justification for top dose:

-Dose-range Finding Test: TA 98, TA 100, TA 1538, TA 1535, TA1537 and the WP2uvrA, both with and without S9-mix: 5, 50, 500, 5000 μg/ plate.

Mutatuion Test 1: TA 98, TA 100, TA 1538, TA 1535, TA1537 and the WP2uvrA, both with and without S9-mix: 312.5, 625, 1250, 2500 and 5000 μg/ plate.

Mutatution Test 2: TA 98, TA 100, TA 1538, TA 1535, TA1537 and the WP2uvrA, both with and without S9-mix: 312.5, 625, 1250, 2500 and 5000 μg/ plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubiity of the material was assessed at 50 mg/mL in water in which it was found to be completely miscible. Water was therefore used as the solvent for this study.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding: 2E+9 cells per mL

DURATION
- Exposure duration: all plates were incubated 3 days

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test material is assessed by applying the following criteria:
a) If treatment with a test material produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of positive dose-relationship, in two separate experiments, with any bacterial strain either in presence or absence of S-9 mix it’s considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b) If treatment with a test material does not produce reproducible increases at least 1.5 times the concurrent controls, at any does level with any bacterial strain, it’s considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
c) If the result obtained fail to satisfy the criteria for a clear “positive” or “negative” response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the material’s mutagenic activity in this test system.
(1) Repeat test may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of narrower dose range that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the material is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(2) If no clear “positive” response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers of any the tester strains were observed following treatment with the test item at any dose level, in the presence or absence of S-9 mix in either mutation test.
Tester strain TA 98 was contaminated in the second mutation test but this contamination did not affect the counting of the plates.

Applicant's summary and conclusion

Conclusions:

When tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of test item, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2 uvrA) were exposed to the test material, diluted in water which was used as a negative control.

 

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induce rats.

 

In the preliminary dose range finding study with dose levels of up to 5000μg/plate no toxicity was observed. A top dose level of 5000μg/plate was chosen for subsequent mutation study. other dose levels used in mutation assays were: 2500, 1250, 625, 312.5μg/plate.

 

No evidence of mutagenic activity was seen at any dose level of test item in either mutation test.

 

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations.

 

It's concluded that, when tested in water, the test item shows no evidence of mutagenic activity in this bacterial system.