Acetoacetamide

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:

22 November 1996 - 13 January 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

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:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetoacetamide

EC Number:

227-774-4

EC Name:

Acetoacetamide

Cas Number:

5977-14-0

Molecular formula:

C4H7NO2

IUPAC Name:

acetoacetamide

Test material form:

solid

Details on test material:

Batch: 2691
white powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and betanaphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Toxicity test: 5000, 1580, 500, 158 and 50 µg/plate
Assay for reverse mutation: 5000, 2500, 1250, 625 and 313 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-aminoanthracene

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity in TA102 at the highest dose-level tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The sterility of the S9 mix and the test substance solutions was confirmed by the absence of colonies on additional agar plates spread separately with these

solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control substances, indicating that the assay

system was functioning correctly.

Applicant's summary and conclusion

Conclusions:

It can be concluded that the test item does not induce reverse mutation in Salmonella Typhimurium under the test conditions.

Executive summary:

The test substance was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic Salmonella typhimurium.

The five tester strains TA1535, TA1537, TA98, TA100 and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

Test substance solutions were prepared using DMSO.

In the toxicity test, the test substance was assayed at a maximum dose-level of 5000 µg/plate and four lower dose-levels spaced at approximately half-log intervals. Slight toxicity, as indicated by thinning of the background lawn, was observed with TA102 tester strain at the highest dose-level tested. The same maximum dose-level was selected for the principal assay.

Two independent experiments were performed, one using a plate incorporation method, the other using a pre-incubation method. The test substance was assayed at a maximum dose-level of 5000 µg/plate and four lower dose-levels, separated by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate.

The test substance did not induce two-fold increases of the number of revertant colonies in the plate-incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence and presence of S9 metabolism.