N,N-dimethyl-3-(octadecyloxy)propylamine

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:

From 31 July 2007 to 27 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Authorities including METI, MHLW and MAFF

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-3-(octadecyloxy)propylamine

EC Number:

241-516-8

EC Name:

N,N-dimethyl-3-(octadecyloxy)propylamine

Cas Number:

17517-01-0

Molecular formula:

C23H49NO

IUPAC Name:

dimethyl[3-(octadecyloxy)propyl]amine

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1 (Range Finding Test): Up to seven concentrations of the test material (ranging between 0.5 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. The dose ranges were allocated as follows:

Salmonella strains, with and without S9-mix: 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
E.coli strain, with and without S9-mix: 50, 150, 500, 1500 and 5000 µg/plate.

Additional dose levels (0.5, 1.5, 5 and 15 µg/plate) were included for the Salmonella strains to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Experiment 2 (Main Test): The second experiment was performed using methodology as described for the range-finding test, using fresh bacterial cultures, test material and control solutions. The test material dose range was 1.5 to 500 µg/plate for all Salmonella strains and 50 to 5000 µg/plate for E.Coli strain WP2uvrA-.

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

vehicle control

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene at 1 µg/plate for TA100, at 2 µg/plate fpr TA1535 and TA1537, at 10 µg/plate for WP2uvrA, at 5 µg/plate for TA98

Details on test system and experimental conditions:

TEST STRAINS
The salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. All of the strains were stored at -196°C in a freezer. Prior to the master strains being used, characterization checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37°C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

MUTATION TEST PROCEDURE
Measured aliquots (0.1ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9 -mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgment about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

The master strains were checked for characteristics, viability and spontaneous reversion rate. The amino acid supplemented top agar and the S)-mix used in both experiments was shown to be sterile.

Results for negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The test material caused a visible reduction in the growth of the bacterial background lawns to all of the Salmonella strains, with and without S9, initially at 150 µg/plate. No toxicity was observed to the E.coli strain, WP2uvrA-. The test material was, therefore, either tested up to the maximum recommended dose level or its toxic limit. A globular precipitate was observed, with the aid of a microscope, at 500 µg/plate becoming visible by eye at 1500 µg/plate. This did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Methods: The test followed the OECD Guideline 471. The Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with solutions of the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.5 and 500 µg/plate depending on strain type. The experiment was repeated on a separate day using a similar dose range as the range-finding test (depending on strain type), fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels (0.5, 1.5, 5 and 15 µg/plate) were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

 

Results: The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial lawns to all of the Salmonella strains, with and without S9, initially at 150 µg/plate. No toxicity was observed to the E. coli strain, WP2uvrA-. The test material was, therefore, either tested up to the maximum recommended dose level or its toxic limit. A globular precipitate was observed, with the aid of a microscope, at 500 µg/plate becoming visible at 1500 µg/plate. This did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

The test material was considered to be non-mutagenic under the conditions of this test.