Fats and Glyceridic oils, vegetable, winterized, reaction products with ammonia-ethanolamine reaction by-products

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:

16-01-2013 to 19-03-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to the guidelines, under GLP

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 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-hexadecyl-4,5-dihydro-1H-imidazol-1-yl)ethyl heptadecanoate; 2-(4-heptadecanoylpiperazin-1-yl)ethyl heptadecanoate; 2-heptadecyl-1-[2-(2-hexadecyl-4,5-dihydro-1H-imidazol-1-yl)ethyl]-4,5-dihydro-1H-imidazole

EC Number:

800-253-4

Cas Number:

1419212-73-9

IUPAC Name:

2-(2-hexadecyl-4,5-dihydro-1H-imidazol-1-yl)ethyl heptadecanoate; 2-(4-heptadecanoylpiperazin-1-yl)ethyl heptadecanoate; 2-heptadecyl-1-[2-(2-hexadecyl-4,5-dihydro-1H-imidazol-1-yl)ethyl]-4,5-dihydro-1H-imidazole

Constituent 2

Reference substance name:

Fats and Glyceridic oils, vegetable, winterized, reaction products with ammonia-ethanolamine reaction by-products

IUPAC Name:

Fats and Glyceridic oils, vegetable, winterized, reaction products with ammonia-ethanolamine reaction by-products

Constituent 3

Reference substance name:

Reaction products of C16 saturated and C18 unsaturated fatty acids and fatty acid glycerides with by-products from 2 aminoethanol and ammonia reaction

IUPAC Name:

Reaction products of C16 saturated and C18 unsaturated fatty acids and fatty acid glycerides with by-products from 2 aminoethanol and ammonia reaction

Constituent 4

Reference substance name:

Fatty acids, C12-18 , reaction products with ethanol, 2-amino, reaction products with ammonia, by products from

IUPAC Name:

Fatty acids, C12-18 , reaction products with ethanol, 2-amino, reaction products with ammonia, by products from

Test material form:

liquid: viscous

Details on test material:

Name of test material (as cited in study report): Fatty acids, C12-18 , reaction products with ethanol, 2-amino, reaction products with ammonia, by products from"
Substance type: UVCB
Purity : 100%
Storage conditions : Room temperature in the dark

Method

Target gene:

Salmonella typhimurium: histidine
E-coli: tryptophan

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:

S9 from rats induced with Phenobarbitone/ß-Naphthoflavone

Test concentrations with justification for top dose:

Salmonella species: 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate with and without metabolic activation in preincubation and plate incorporation assay
E. coli: 50, 150, 500, 1500 and 5000 ug/plate with and without metabolic activation in plate incorporation assay; 15, 50, 150, 500, 1500 and 5000 ug/plate with and without metabolic activation in preincubation assay

Vehicle / solvent:

acetone (100 mg/mL)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

for TA100, TA1535 and WP2uvrA without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

for TA1537 without metabolic activation

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

for TA100, TA1535, TA 1537 and WP2uvrA with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

for TA98 without metabolic acivation

Positive control substance:

4-nitroquinoline-N-oxide

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

for TA98 with metabolic activation

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: 1st test in agar (plate incorporation); 2nd test preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth rate + presence of bacterial background lawn

COLONY COUNT:
Colony counter

Evaluation criteria:

A test item will be considered mutagenic (positive) in the test system if one or more of these criteria are met
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

Statistics:

Not specified according to UKEMS

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:

reduction of bacterial background lawn at 1500 ug/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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

presence of greasy precipitate did not hinder the evaluation at 1500 and 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results
negative without metabolic activation
negative with metabolic activation

The test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.

Executive summary:

The test substance was tested in an Ames test at concentrations of 1.5 to 1500 ug/plate (cytotoxicity obeserved) in the salmonella strains TA98, TA100, TA1535 and TA1537. The concentrations in the test on E. coli WP2 uvr A were 15 -5000 ug/plate. At 1500 and 5000 ug/plate a greasy precipitate was observed, that did not hamper the evaluation. In a plate incorporation and a pre-incubation assay (both performed in triplicate) with and without metabolic activation, the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr.