Fatty acids, C16 and C18-20 (even numbered, unsaturated), 2-ethylhexyl esters

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:

24 November 2004 to 10 December 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

In the pre-incubation assay 50 µL test solution /solvent control, or 100 µL positive control, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates. In the more sensitive pre-incubation assay ethanol is toxic to the bacteria. Therefore, a lower amount of test solution was used.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP)

IUPAC Name:

Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP)

Details on test material:

- Name of test material (as cited in study report): Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP)

- Physical state: yellowish liquid
- Analytical purity: 100%

- Purity test date:Not stated
- Lot/batch No.: 463411
- Expiration date of the lot/batch: 15 January 2008

- Stability under test conditions: stable
- Storage condition of test material: room temp.

Method

Target gene:

The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- to his+ and trp- to trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no

Additional strain / cell type characteristics:

other: histidine independence

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: tryptophan dependence

Metabolic activation:

with and without

Metabolic activation system:

liver mitochondrial S-9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. This was reported as Experiment I since no toxic effects were observed and the maximum concentration was selected for the high dose, 5000 µg/plate.
The following concentrations were tested:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) was dissolved in ethanol (purity > 99.8 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria .
The test item precipitated in the overlay agar with and without S9 mix at 5000 µg/plate in Experiment I and from 2500 µg/plate up to 5000 µg/plate in Experiment II. The undissolved particles had no influence on the data recording.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

solvent control - ethanol

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: without S-9, for strains TA1535 and TA100

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene diamine without S-9, for strains TA1537 and TA98

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: For E.coli WP2 uvrA without S-9

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, with S-9, all tester strains

Details on test system and experimental conditions:

For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

In the pre-incubation assay 50 µL test solution /solvent control, or 100 µL positive control, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark

Evaluation criteria:

Acceptability criteria
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
-the positive control substances should produce a significant increase in mutant colony
frequencies

Evaluation criteria
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment

Statistics:

No statistical evaluation of the data was required

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:

no cytotoxicity, but tested up to precipitating concentrations

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) was assessed for its potential to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments, with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Tables of results for plate incorporation and pre-incubation test results are attached as word files

Applicant's summary and conclusion

Conclusions:

During this mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains of S. typhimurium and E.coli used.
Therefore, Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation.

Each concentration, including the controls, was tested in triplicate.

Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP)

was tested at the following concentrations:

Experiment I:              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:             33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Relevant historical control data were also presented in the sudy report but are not reproduced in this summary.