Fatty acids, tall-oil, 1-methyl-1,2-ethanediyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (cofactor-supplemented liver fraction of phenobarbital/5,6-benzoflavone-induced rats)

Test concentrations with justification for top dose:

Experiment 1: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate
Experiment 2: 0; 50; 150; 500; 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: A solution suitable for use was obtained in ethanol at a concentration of 50 mg/mL (to provide a final concentration of 5000 μg/plate, the maximum guideline recommended test substance concentration).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used in tests without metabolic activation for strains TA100 and TA1535 at 2 µg/plate.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used in tests without metabolic activation for strain TA1537 at 50 µg/plate.

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used in tests without metabolic activation for strain TA98 at 2 µg/plate.

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Used in tests without metabolic activation for strain WP2 uvrA (pKM101) at 2 µg/plate.

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used in tests with metabolic activation for strains TA100 and TA1535 at 5 µg/plate; for strain WP2 uvrA (pKM101) at 10 µg/plate.

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Used in tests with metabolic activation for strains TA98 and TA1537 at 5 µg/plate.

Details on test system and experimental conditions:

Two independent standard plate incorporation assays were performed with and without metabolic activation (S9 mix). Test procedures varied in the proportion of S9 content in the S9 mix (10% v/v and 20% v/v in Experiment 1 and 2, respectively).

3 Pleates per treatment were incubated at approx. 37 °C for ca. 72 h. The appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter.

Cytotoxiciy was defined as a substantial reduction in mean revertant colony counts and/or by a sparse or absent background bacterial lawn.

Evaluation criteria:

The test material is considered to exhibit mutagenic activity in this assay if a reproducible increase in revertant colony numbers of at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship occurs.

A test substance is considered non-mutagenic in this test if exposure to the test material does not produce a reproducible increase in revertant colony numbers.

Statistics:

The data were not statistically analysed. The study result was unequivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No evidence of toxicity was obtained.
At a test material concentration of 5000 µg/plate, precipitate was observed in all treatments.
All sterility control plates were colony free. Hence the absence of microbial contamination of the S9 mix, buffer and test material formulation was confirmed. Viability counts met the acceptance criteria. See tables on results and viability attached as background material

Applicant's summary and conclusion

Conclusions:

no mutagenicity observed in this Ames test