N-[3-(dimethylamino)propyl]oleamide

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: Study conducted in accordance with OECD guideline 476 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]oleamide

EC Number:

203-661-5

EC Name:

N-[3-(dimethylamino)propyl]oleamide

Cas Number:

109-28-4

Molecular formula:

C23H46N2O

IUPAC Name:

(9Z)-N-[3-(dimethylamino)propyl]octadec-9-enamide

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 3-Dimethyl-aminopropyl-ölsäureamide
- Betach: R401/57
- Purity: 78.2%
- Color: reddish
- Test substance stability: Stability of the test substance at room temperature in water for 4 hours and 7 days was determined analytically.

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from male Sprague-Dawley rats, induction by a single i.p. injection of 500 mg/kg body weight Aroclor 1254 in corn oil 5 days before sacrifice.

Test concentrations with justification for top dose:

Experiment 1: 0, 0.16, 0.63, 1.25, 2.5, 5.0, 10.0, 20.0 μg/ml
Experiment 2: 0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 μg/ml (without S9 mix); 0, 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0 μg/ml (with S9 mix)

Vehicle / solvent:

Ham's F12 culture medium

Controls

Negative solvent / vehicle controls:

yes

Remarks:

The negative control cultures were treated in parallel to the other treatment groups, but only culture medium without test substance was used.

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Remarks:

EMS (without metabolic activation): 300 μg/ml, dissolved in Ham's F12 medium without FCS; 3-MCA (with metabolic activation): 10 μg/ml, dissolved in DMSO, diluted with Ham's F12 medium without FCS.

Details on test system and experimental conditions:

- A pretest for toxicity was performed following the method described for the main experiment. In the pretest, concentrations between 20 and 5000 μg/ml were applied both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. Precipitation occured at 78 μg/ml and above (with and without S9 mix). Strong cytotoxicity was observed at all concentrations in the pretest after 4 hours and 24 hours treatment with and without S9 mix (relative cloning efficiency reduced to under 20% relative survival).
- Pretreatment of cells: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
- Method of test substance application: in medium
- Application of test substance: 20-24 hours after seeding
- Incubation time: 4 hours
- Expression period: 6-8 days
- Selection period: 6-7 days

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- Absolute cloning efficiencies of the negative controls should be >=50% (with and without S9 mix).
- Background mutant frequency in the negative controls should fall within historical negative control data range.
- The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies in the range of historical positive control data.
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. 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 of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

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 the range of historical negative control data.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong cytotoxicity was observed at 20 μg/ml and above.

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions chosen, 3-Dimethyl-aminopropyl-ölsäureamide is not mutagenic in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The substance 3-Dimethyl-aminopropyl-ölsäureamide was tested for its ability to induce gene mutations at the HPRT locus in CHO cells in vitro. Two independent experiments were carried out with and without Aroclor-induced rat liver S9 mix, using test substance concentrations of 0-20 μg/ml (with and without S9 mix) in experiment 1, as well as 0-6 μg/ml (without S9 mix) and 0-20 μg/ml (with S9 mix) in experiment 2. A pretest for toxicity showed strong cytotoxicity at 20 μg/ml and above.

After a 20-24 hour attachment period, the cells were treated with the test substance for 4 hours, followed by an expression phase of 6 -8 days and a selection period in medium containing 10 μg/ml 6-thioguanine of about 1 week. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. It can be concluded that under the experimental conditions chosen, 3-Dimethyl-aminopropyl-ölsäureamide is not mutagenic in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.