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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Density: 0.94 g/cm³
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: 41334368E0

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 mix: liver from male Wistar rats (induced with phenobarbital and ß-naphthoflavone)
Test concentrations with justification for top dose:
Experiment I + II: 31.6; 100; 316; 1000; 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua dest.

Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments

TREATMENT AND HARVEST SCHEDULE:
Standard plate test (Experiment II):
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control)
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation)
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain)
2000 µL Overlay agar

Preincubatintest (Experiment II):
For the pre-incubation method 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37°C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate. For each strain and dose level, including the controls, three plates were used. After solidification the plates were inverted and incubated at 37°C for at least 48 h in the dark.

Evaluation criteria:
The mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the numer of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows.
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higer than the reversion rate of the solvent control.

Results and discussion

Test results
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537, TA 102
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 precipitation of the test item was observed in any tester strain usen in experiment I and II (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numers of any of the five tester strains were observed follwoing treatment with N,N-Dimethylacetamide at any concentratin level, neither in the presence nor absence of metabolic activation in experiment I and II. The references mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, N,N-Dimethylacetamide did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, N,N-Dimethylacetamide is considered to be non-mutagenic in theis bacterial reverse mutation assay.