N-[3-(dimethylamino)propyl]stearamide

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:

1995-05-23 to 1995-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[3-(dimethylamino)propyl]stearamide

EC Number:

231-609-1

EC Name:

N-[3-(dimethylamino)propyl]stearamide

Cas Number:

7651-02-7

Molecular formula:

C23H48N2O

IUPAC Name:

N-[3-(dimethylamino)propyl]octadecanamide

Details on test material:

- chemical name: Stearic acid 3-(dimethylaminopropyl)amide

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix (Aroclor 1254-induced rat liver S-9 plus cofactors)

Test concentrations with justification for top dose:

Range finding test with strains S. typhimurium TA 100 and E.coli WP2uvrA: 5, 10, 50, 100, 500, 1000, 5000 µg/plate with and without mammalian metabolic activation
Main study (1st and 2nd mutation assay): S. typhimurium strains: 5, 10, 25, 50, 75 µg/plate without mammalian metabolic activation; 25, 50, 75, 100, 250 µg/plate with mammalian metabolic activation
E. coli strain WP2uvrA: 10, 25, 50, 75, 100 µg/plate without mammalian metabolic activation; 50, 75, 100, 250, 500 µg/plate with mammalian metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water; source: Sitek, Lot-No: 022895 and 060195
- Justification for choice of solvent/vehicle: The stock solutions of the test article were prepared in water. Therefore, water was used as the solvent control.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: (without metabolic activation)

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st mutation assay: plate incorporation; 2nd mutation assay: preincuation method (to confirm the first assay)

DURATION (plate incorporation)
- Preincubation period: 68 h

DURATION (preincubation)
- Preincubation period: 20 min
- Exposure duration: 68 h

NUMBER OF REPLICATIONS: 3

SELECTION AGENT (mutation assays): all Salmonella typhimurium strains: histidine, rfa mutation; E. coli strain: tryptophane. Strains TA 98 and TA100 were tested in addition for the presence of pKM101 plasmid.


DETERMINATION OF CYTOTOXICITY
- Method: reduction of the background lawn, viability or reduction of the spontaneous reversion rate

RANGE FINDING TEST
- In order to determine the toxicity and to select the appropriate test article concentrations for the Mutation Assay,
a Range Finding Test was performed using strains TA100 and WP2uvrA.
- Seven doses of test article, ranging from 5.0 to 5000 µg/plate, were evaluated with and without Aroclor-induced rat liver S-9, using one plate per dose.

Evaluation criteria:

Criteria for a Negative Response.
A response is considered to have caused a negative response if all of the strains treated with the test article have mean reversion frequencies that are no greater than twice that of the mean reversion frequencies of the corresponding solvent control plates in TA98 and TA100 and no greater than three times in TA1535, TA1537 and WP2uvrA, and there is no evidence of a dose dependent response.
Criteria for a Positive Response.
A response is considered to have caused a positive response if either strain TA98 or TA100 has a dose that produces a mean reversion frequency that is two times or more greater than the mean reversion frequency of the corresponding solvent control plates or if either strain TA1535, TA1537 or WP2uvrA has a dose producing a three-fold increase in the mean reversion frequency compared to the solvent control frequency. In addition, the response must be dose dependent or increasing concentrations of the extract must show increasing mean reversion frequencies. In evaluating the results, consideration will be given to the degree of toxicity exhibited by the dose causing the two-fold/three-fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency.
Criteria for an Equivocal Response.
A response is considered to have caused an equivocal response if it does not fulfill the criteria of either a negative or a positive response and/or the Study Director does not consider the response to be either positive or negative.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>/= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitate at any concentration with and without activation
- Comparison of the solvent control values with those for test substance-treated plates for the first Mutation Assay indicated that the test substance produced a negative response in the presence and absence of S-9 activation. All of the strains treated with the test substance exhibited a mean reversion frequency that was similar to the corresponding solvent control, and there was no evidence of a dose-dependent response.
- Since the results of the first Mutation Assay were negative, the preincubation method of exposure was used.
- 2nd assay with activation: confirmation of results of 1st assay
- 2nd assay without activation: all of the S. typhimurium strains showed signs of toxicity at the highest concentrations of 50 and 75 µg/plate. There were varying degrees of toxicity in the different Salmonella strains. Toxicity ranged from a noticeable thinning of the microcolony lawn compared to solvent control to an absence of any microcolonies. There was an increase in the microcolony size due to toxicity, thus giving a higher count for the revertants with the automatic colony counter. All of the lower concentrations had a similar number of revertants as in the corresponding solvent controls and there was no dose-related response. Thus the test substance produced a negative response in the confirmatory assay.

RANGE-FINDING/SCREENING STUDIES:
- Strain TA 100 without activation: toxic responses at 100, 500, 1000, and 5000 µg/plate. There were no colonies in the viable count plates. The concentration of 50 µg/plate had a 7% relative cloning efficency (RCE). There was marked reduction in the microcolony lawn at 100 µg/plate and an absence of microcolony lawn at 500 µg/plate and above. There was a reduction in the number of revertants at these concentrations, indicating that these concentrations were toxic. The remaining lower concentrations were nontoxic.
- Strain TA 100 with activation: toxic responses at 500, 1000 and 5000 µg/plate. There were no colonies in the viable count plates at the highest two concentrations of 1000 and 5000 µg/plate. The RCE was only 2% at 500 µg/plate. There was a marked reduction in the microcolony lawn at 500 µg/plate and above. The number of revertants was also depressed in the highest two concentrations of 1000 and 5000 µg/mL. The remaining concentrations were nontoxic.
- Strain WP2uvrA without activation: signs of toxicity at the highest three concentrations of 500, 1000 and 5000 µg/plate. There were no colonies in the viable count plates at 1000 and 5000 µg/plate and 500 µg/plate had an RCE of 1%. There was an extreme reduction in microcolony lawn at 500 µg/plate and absence of microcolony at 1000 and 5000 µg/plate. There was a reduction in the number of revertants in these two concentrations. The
remaining lower concentrations were nontoxic.
- Strain WP2uvrA with activation: signs of extreme toxicity with the highest two concentrations of 1000 and 5000 µg/plate. 1000 µg/plate had an RCE of 13%. There was an absence of microcolony lawn in both of these concentrations and no revertants were present. The 500 µg/plate had a reduction in the number of revertants. The remaining lower concentrations were basically nontoxic.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Stearic acid 3-(dimethylaminopropyl)amide (a.i. 85%) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria equivalent to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed to Stearic acid 3-(dimethylaminopropyl)amide (a.i. 85 %) at concentrations of 5, 10, 25, 50, and 75 µg/plate in the absence of mammalian metabolic activation, and at concentrations of 25, 50, 75, 100 and 250 µg/plate in the presence of mammalian metabolic activation. Strain E. coli WP2uvrA was tested at concentrations of 10, 25, 50, 75, and 100 µg/plate in the absence and at 50, 75, 100, 250, and 500µg/plate in the presence of metabolic mammalian activation.

Two tests were performed, the first test using the plate incorporation and the second test the preincubation method.

 

In the first mutation assay (plate incorporation) Stearic acid 3-(dimethylaminopropyl)amide did not induce a significant dose-related increase in the number of revertant colonies in all five tested strains, both in the absence and presence of mammalian metabolic activation. These results were confirmed in the second mutation assay (preincubation method). No precipitation was observed.

Cytotoxic effects of the test substance were observed in the preincubation assay in all S. typhimurium strains, at a concentration of >/= 50 µg/plate in the absence of mammalian metabolic activation. Additionally at range finding test cytotoxic effects were shown at >/= 500 µg/plate in the E. coli. WP2 uvr A strain.

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed.

 

There was no evidence of induced mutant colonies over background.

 

Based on the results of this study it is concluded that Stearic acid 3-(dimethylaminopropyl)amide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

This study is classified as acceptable. It satisfies the requirements for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.