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Toxicological information

Genetic toxicity: in vitro

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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: GLP compliant study conducted in accordance with international guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyl-3-oxobutyramide
EC Number:
218-059-8
EC Name:
N,N-dimethyl-3-oxobutyramide
Cas Number:
2044-64-6
Molecular formula:
C6H11NO2
IUPAC Name:
N,N-dimethyl-3-oxobutyramide
Constituent 2
Reference substance name:
N,N-Dimethylacetoacetamide
IUPAC Name:
N,N-Dimethylacetoacetamide
Test material form:
other: yellow liquid
Details on test material:
Test item name: N,N-Dimethylacetoacetamide

Method

Target gene:
Evaluation of the test substance for its ability to induce reverse mutations either in the presence or absence of an exogenous S9 metabolic
activation system at the histidine locus in the genome of Salmonella typhimurium (strains TA98, TA100, TA1535, and TA1537),
and at the tryptophan locus of the Escherichia coli strain WP2 uvrA.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 from male Sprague-Dawley rats
Test concentrations with justification for top dose:
33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate
Vehicle / solvent:
HPLC-grade sterile water
Controls
Untreated negative controls:
no
Remarks:
Concurrent solvent control only
Negative solvent / vehicle controls:
yes
Remarks:
HPLC-grade sterile water
True negative controls:
no
Remarks:
Concurrent solvent control only
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: acridine mutagen ICR-191, 2-aminoanthracene
Details on test system and experimental conditions:
Test Solution:
------------
The positive controls were dissolved in dimethyl sulfoxide (DMSO, CAS# 67-68-6, 99.9% purity, EMD), except for the test item, sodium azide and ICR-191, which were dissolved in sterile water.

Preparation of the Metabolic Activation System:
------------------------------------------
Liver homogenate (S9, average protein concentration: 37.8 ± 1.0 mg/mL) prepared from male Sprague-Dawley rats induced with Aroclor 1254
was purchased commercially (Moltox Inc., Boone, North Carolina).
The S9 was thawed and the 10% S9 mix prepared immediately prior to its use. The S9 mix was held on ice at all times.

Preparation and Storage of Tester Strain:
------------------------------------
Frozen permanent stocks of all tester strains were prepared by growing fresh overnight cultures with the addition of 0.09 mL DMSO per
milliliter of culture. Aliquots were frozen in dry ice and stored at ≤-70°C.
Master plates were prepared by streaking each tester strain from a frozen permanent stock onto either nutrient agar plates or minimal
glucose agar plates. The minimal glucose agar plates were supplemented with either histidine and biotin or tryptophan, and for strains
containing the pKM101 plasmid, ampicillin. Tester strain master plates were stored at 5 ± 3°C and assigned a one-month expiration date.
Overnight cultures for use in the study were inoculated from the appropriate master plates. Cultures were placed in a shaker/incubator for
overnight at 150 ± 50 rpm and 37 ± 2°C. To ensure that appropriate numbers of bacteria are plated, the length of incubation was determined
by spectrophotometric monitoring of culture density. Cultures were harvested when the tester strain culture titers were equal to or greater
than 0.3 x 109 cells per milliliter.




Evaluation criteria:
Evaluation:
----------
Criteria for a positive response:
1. Strains TA1535 and TA1537
Data will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 3.0-fold the mean concurrent negative
control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response
associated with increasing concentrations of the test substance.
2. Strains TA98, TA100 and WP2uvrA
Data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 2.0-fold the mean concurrent
negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response to
increasing concentrations of the test substance.
I. Data Presentation
Individual plate counts for all treated, positive and negative controls, and an evaluation of the bacterial background lawn are reported.
For each tester strain, the mean of the number of revertants and the standard deviations were calculated.
Statistics:
An analytical verification of the test substance concentrations was not conducted.

Results and discussion

Test resultsopen allclose all
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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:
not examined
Positive controls validity:
valid
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
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity Test:
------------
No toxicity or test substance precipitate was observed at any dose level with any tester strain in the presence or absence of
S9 metabolic activation.

Any other information on results incl. tables

Sterility Controls:

No contaminant colonies were observed on the sterility plates for the most concentrated test substance dilution (50 mg/mL) and the S9 and sham mixes.

Solubility:

The test substance was soluble in water at 50 mg/mL, the highest concentration that was tested in the study.

Toxicity-Mutation Test:

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in the test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 μg/plate. No positive mutagenic responses were observed at any dose level in any tester strain in the presence or absence of S9 metabolic activation. No toxicity or test substance precipitate was observed at any dose level with any tester strain in the presence or absence of S9 metabolic activation.

Mutagenicity Test

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the presence and absence of S9. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in the test were 333, 667, 1000, 3333, and 5000 μg/plate for all tester strains. No positive mutagenic responses were observed at any dose level or with any tester strain in either the presence or the absence of S9. No toxicity or test substance precipitate was observed at any dose level with any tester strain in either the presence or the absence of S9.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

All criteria for a valid study were met. Under the conditions of this study, DMAA showed no evidence of mutagenicity in the Bacterial Reverse
Mutation Test either in the presence or absence of Aroclor-induced rat liver S9. The test substance was concluded to be negative in this
study.
Executive summary:

A study according to OECD Guideline 471 (Bacterial Reverse Mutation Assay), EU Method B.13/14 and EPA OPPTS 870.5100 was carried out in year 2005. Under the conditions of this study, DMAA showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the presence or absence of Aroclor-induced rat liver S9. The test substance was concluded to be negative in this study.