trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline

EC Number:

255-490-0

EC Name:

trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline

Cas Number:

41672-81-5

Molecular formula:

C37H69NO5

IUPAC Name:

1-palmitoyl-4-(palmitoyloxy)-L-proline

Details on test material:

- chemical name: palmitoyl-1 palmitoyloxy-4-proline
- form: white solid
- storage conditions: room temperature in the dark
- batch number: 97275001

Method

Target gene:

Mutation in Histidine biosynthesis for Salmonella typhimurium: his D 3052/strain TA98 (frameshift), his G 46/strains TA100 and TA1535 (base-pair substitution) and his C 3076/strain TA1537 (frameshift)
Mutation in Tryptophan biosynthesis for Escherichia coli : WP2uvrA- (deletion in an excision repair gene)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/well

Vehicle / solvent:

dimethyl formamide

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for TA100, TA1535 and WP2uvrA-strains without S9 activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA1537 strain without S9 activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

for TA98 strain without S9 activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for TA100, TA1535, TA1537 and WP2uvrA- with S9 activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethylformamide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

for TA98 with S9 activation

Details on test system and experimental conditions:

(1) Preliminary evaluation of cytotoxicity of the substance
The cytotoxicity was performed of the Salmonella Typhimurium strain TA 100 and in the Escherichia Coli WP2uvrA- with and without metabolic activation (S9 mix). The substance was tested at 10 concentrations : 0.15 ; 0.5 ; 1.5 ; 5 ; 15; 50; 150 ; 500 ; 1500 and 5000 µg/dish. A vehicle control, dimethyl formamide was added. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was plated onto a sterile plates of Vogel-Bonner Minimal agar. The Petri dishes were incubated at 37°C for 48 hours.
(2) Main study
4 strains of Salmonella Typhimurium were tested: TA98, TA100, TA1535 and TA1537. 1 strain of Escherichia Coli was tested : WP2uvrA-
As the concentration of 5 mg/plate was not toxic in the strain TA 100 and UWP2uvrA- with and without metabolic activation during the preliminary
study, the following concentrations were tested during the main study: 5, 15, 50 ; 150 ; 500; 1500 and 5000 µg/dish.
Each concentration was tested 3 times under a constant volume of 0.1 ml with and without metabolic activation (S9 fraction obtained from rat livers pre-treated with phenobarbitone/b-naphthoflavone at 80-100 mg/kg/day, 3 days) and used at on each strain. The incubation system contained 0.1 ml of the diluted substance + 2 ml of top agar + 0.1 ml of bacterial culture +0.5 ml of S9 mix or phosphate buffer. The mixture was plated onto the surface of Vogel-Bonner Minimal agar plate and incubated at 37°C for 48 hours.
The vehicle, imethyl formamide, and positive controls are included in the study. Positive controls are : 4-Nitroquinoline-1-oxide at 0.2 µg/plate for TA-98; N-ethyl-N'-nitro-nitroguanidine at 3 µg/plate for TA-100, at 5 µg/plate for TA-1535 and at 2 µg/plate for WP2uvrA-; 9-aminoacridine at 80 µg/plate for TA-1537. 2-aminoanthracene was used as positive reference, in presence of S9 mix, at 1 µg/plate for TA100 ; at 2 µg/plate for TA1535 and TA1537 and at 10 µg/plate for WP2uvrA-. Benzo(a)pyrene was used, in presence of S9 mix, at 5 µg/plate for TA98.
All the results were confirmed in a second study independent from the first. For the second experiment, the test product was tested at 50, 150, 500, 1500 and 5000 µg/plate for the strains TA98 and WPEuvrA-. For the strains TA100, TA1535 and TA1537, the test product was tested at 5 to 5000 µg/plate like in experiment 1.

Evaluation criteria:

The test material may considered to be positive in the test system if it should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria. If a greater than twofold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Statistics:

calculation of the mean and standard deviation of n=3 per study.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2

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

Test results 2

Species / strain:

S. typhimurium TA 100

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium TA 1537

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

Test results 5

Species / strain:

S. typhimurium TA 1535

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:

In the preliminary toxicity study performed in the strains TA100, WP2uvrA-, a precipitation of the product was observed at 5000 µg/plate. The first indication of toxic response was observed at 500 µg/plate. The test material, was therefore, tested up to the maximum recommended dose of 5000 µg/plate or of the toxic limit, depending on bacterial strain.
In the main study, the test substance caused a visible reduction in the growth of the bacterial lawn and/or a reduction in the frequency of revertant colonies in all of the Salmonella strains both with and without metabolic activation at 500 µg/plate.
under experimental conditions employed, no value obtained in the presence of the test article was greater than or equal to twice the value obtained in the presence of the vehicle with and without metabolic activation on the bacterial strains used.
All the results obtained in presence of positive controls were significant with and without metabolic activation in the used bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The substance was tested on 4 strains of salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in 1 strain of Escherichia Coli (WP2uvrA-), with or without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strains TA-100 and WP2uvrA- with and without metabolic activation.

The 7 concentrations (5 - 15 -50 - 150 - 500 - 1500 and 5000 µg/plate) were tested 3 times on the 5 strains mentioned above with and without metabolic activation. The results were confirmed in a second study, independant of the first.

In each study was included a negative control (vehicle = dimethyl formamide) and a positive control (specific standard mutagen).

The test substance caused a visible reduction in the growth of the bacterial lawn and/or a reduction in the frequency of revertant colonies in all of the Salmonella strains both with and without metabolic activation. The first indication of toxic response was observed at 500 µg/plate. The test material, was therefore, tested up to the maximum recommended dose of 5000 µg/plate or of the toxic limit, depending on bacterial strain.

Under the experimental conditions employed, the test substance did not show any mutagenic potential for the strain TA98, TA100, TA1535, TA1537 and WP2uvrA- with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:

The test article, is not mutagenic as it induces no significant increase in the number of revertants with and without metabolic activation of Salmonella Typhimurium TA98, TA100, TA1535 and TA1537 and in Escherichia Coli WP2uvrA-.

Executive summary:

The substance was tested on 4 strains of salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in 1 strain of Escherichia Coli (WP2uvrA-), with or without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strains TA-100 and WP2uvrA- with and without metabolic activation.

The 7 concentrations (5 - 15 -50 - 150 - 500 - 1500 and 5000 µg/plate) were tested 3 times on the 5 strains mentioned above with and without metabolic activation. The results were confirmed in a second study, independant of the first.

In each study was included a negative control (vehicle = dimethyl formamide) and a positive control (specific standard mutagen).

The test substance caused a visible reduction in the growth of the bacterial lawn and/or a reduction in the frequency of revertant colonies in all of the Salmonella strains both with and without metabolic activation. The first indication of toxic response was observed at 500 µg/plate. The test material, was therefore, tested up to the maximum recommended dose of 5000 µg/plate or of the toxic limit, depending on bacterial strain.

Under the experimental conditions employed, the test substance did not show any mutagenic potential for the strain TA98, TA100, TA1535, TA1537 and WP2uvrA- with and without metabolic activation.