N,N-dimethylacrylamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylacrylamide

EC Number:

220-237-5

EC Name:

N,N-dimethylacrylamide

Cas Number:

2680-03-7

Molecular formula:

C5H9NO

IUPAC Name:

N,N-dimethylacrylamide

Details on test material:

- Name of test material (as cited in study report): N,N-Dimethylacrylamide
- Test substance No.: 11/0599-2
- Molecular weight: 99.13 g/mol
- Physical state: Liquid, colorless to yellowish, clear
- Purity: 99.4 corr. area % (see analytical report, study code 12L00359)
- Lot/batch No.: 20120615
- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations
Storage stability: The stability of the test substance under storage conditions was guaranteed until 15 Jun 2013 as indicated by the sponsor.
- Storage condition of test material: Room temperature; protected from light

Method

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st experiment
125.0, 250.0, 500.0, 1000.0 µg/mL (4-h exposure, with and without S9 mix)

2nd experiment
62.5, 125.0, 250.0, 500.0, 1000.0 µg/mL (24-h exposure, without S9 mix)
175.0, 350.0, 700.0, 1000.0 µg/mL (4-h exposure, with S9 mix)

Vehicle / solvent:

medium (Ham's F12)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20-24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression time (cells in growth medium): The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).
- Selection time (if incubation with a selection agent): For selection of the mutants, six 75 cm2 flasks with 3x10^5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

NUMBER OF CELLS EVALUATED: The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within the historical negative control data range of 0.00 -16.43 mutants per 10^6 clonable cells.
- The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data).
- At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
- Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
- Evidence of the reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
In the pretest for toxicity based on the purity and the molecular weight of the test substance 1 000 μg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pretest the parameters pH value and osmolarity were not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no precipitation of the test substance in culture medium was observed up to the highest required concentration of 1 000 μg/mL in the absence and presence of S9 mix. No cytotoxicity as indicated by a reduced relative cloning efficiency of about or below 20% relative survival was observed under all test conditions up to the highest applied concentration.

MUTANT FREQUENCY
No relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 1.07 – 6.92 per 10^6 cells) were close to the respective vehicle control values (MFcorr.: 1.88 – 6.45 per 10^6 cells) and clearly within the range of the historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 10^6 cells; with S9 mix: MFcorr.: 0.00 – 15.83 per 10^6 cells).
The positive control substances EMS (without S9 mix; 300 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 126.53 – 516.00 per 10^6 cells; with S9 mix: MFcorr.: 207.01 – 256.65 per 10^6 cells) were clearly within the historical positive control data range (without S9 mix: MFcorr.: 47.35 – 1 338.10 per 10^6 cells; with S9 mix: MFcorr.: 131.35 – 1 250.00 per 10^6 cells).

CYTOTOXICITY
Cytotoxic effects as indicated by clearly reduced cloning efficiencies of about or below 20% relative survival were not observed in the 1st and 2nd Experiment in the absence and presence of S9 mix up to the highest required concentration. The cell densities were not distinctly reduced at 1st subculture at any experimental condition.

CELL MORPHOLOGY
There were no adverse observations on cell morphology (cell attachment).

TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest required test substance concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.