Fatty acids, tall-oil, maleated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

of 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

of 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-18 and C16-18-unsatd., maleated

EC Number:

288-306-2

EC Name:

Fatty acids, C14-18 and C16-18-unsatd., maleated

Cas Number:

85711-46-2

IUPAC Name:

85711-46-2

Method

Target gene:

TK

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-5, RPMI-10 and RPMI-20 containing 5% v/v, 10% v/v and 20% v/v heat inactivated horse serum, respectively
and each medium containing: Antibiotic-antimycotic solution (containing penicillin, streptomycin & amphotericin-B),
Pyruvic acid and
NaHCO3.
RPMI-5 and RPMI-10 additionally contained Pluronic-F68.

- Type and identity of media, in general used for cell culture: RPMI-10
- Type of media used for Treatment media: RPMI-5, culture incubated for 3 or 24 hours at 37± 1 °C (approximately 5% CO2 in air)
- Plating for survival: Dilution with RPMI-10 followed by dilution with RPMI-20
- 3-day mutation expression period: RPMI-10
- Plating for viability after the expression period: Dilution with RPMI-10 followed by dilution with RPMI-20

Management of mouse lymphoma cell line L5178Y TK+/- 3.7.2 C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Wistar rats treated with phenobarbital and beta-naphthoflavone (80 mg/kg/day over 3 consecutive days) for enzyme induction. Final concentration of liver homogenate in the test system was 2%.

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (plating efficiency relative to that of vehicle controls)
Test concentrations at 3 h exposure with (+S9) and without (–S9) metabolic activation and at 24 h exposure without metabolic activation (–S9):
39.06, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 12.5, 25, 50, 75, 100, 112.5, 125, 137.5, 150 and 200 μg/mL
Mutant phenotype determination at: 12.5, 25, 50, 75, 100, 112.5, 125, 137.5 and 150 μg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 12.5, 25, 50, 100, 150, 200, 250, 300 and 400 μg/mL
Mutant phenotype determination at: 12.5, 25, 50, 100 and 150 μg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 6.25, 12.5, 25, 50, 75, 100, 112.5, 125, 137.5, 150 and 200 μg/mL
Mutant phenotype determination at: 6.25, 12.5, 25, 50, 75, 100, 112.5, 125 and 137.5 μg/mL

Experiment 2, 3 h exposure (+S9):
Exposure concentrations: 12.5, 25, 50, 100, 120, 140, 160, 180, 200 and 250 μg/mL
Mutant phenotype determination at: 12.5, 25, 50, 100, 120, 140 and 160 μg/mL

Experiment 3, 24 h exposure (–S9):
Exposure concentrations: 25, 50, 75, 100, 110, 120, 130, 140, 150, 160 and 170 μg/mL
Mutant phenotype determination at: 25, 50, 75, 100 and 110 μg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
Fluctuation in osmolality and pH between treated groups and the vehicle control group should not exceed 50 mOsm/kg and 0.5, respectively. The presence of precipitate should not interfere with mutant phenotype determination. In addition, for a toxic test material, at least 4 analysable concentrations should be achieved which ideally span the toxicity range of 100 to 10-20% harmonised relative survival.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Justification for choice of solvent/vehicle:
DMSO was chosen as a vehicle, because it is compatible with the test system. In a preliminary solubility trial a suitable dilution of WS400104 in DMSO was achieved at 250 mg/mL. This concentration produced test material concentrations in culture medium of 2500 and 5000 µg/mL when administering the DMSO test material dilution to the culture medium at 1% v/v and 2% v/v, respectively. Adequate miscibility and/or solubility of the test material in the compatible vehicle facilitates maximum exposure of the cells in the test system to the test material.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Remarks:

0.15 µg/mL for 3 h treatment, 0.10 µg/mL for 24 h treatment, vehicle DMSO

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control substance for tests without metabolic activation (-S9) in Experiments 1, 2 and 3

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (1% v/v final concentration in the medium)

True negative controls:

no

Positive controls:

yes

Remarks:

4 µg/mL, vehicle DMSO

Positive control substance:

cyclophosphamide

Remarks:

Positive control substance for tests with metabolic activation (+S9) in Experiments 1 and 2

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: Experiment 1: 3 h exposure with (+S9) and without (–S9) metabolic activation
Experiment 2: 3 h exposure with (+S9) and 24 h exposure without metabolic activation (–S9)
Experiment 3: 24 h exposure without metabolic activation (–S9)

- Selection time: Approximately 3 days after the end of exposure addition of the selection agent trifluorothymidine (TFT)
then allowing approximately 2 weeks for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT), at 3 µg/mL in final medium

NUMBER OF REPLICATIONS: 2 cultures at each test concentration, negative control, vehicle control or positive control
- from each test concentration, negative, vehicle or positive control (per culture):
2 flasks for assessment of growth in suspension,
two 96-well plates for assessment of survival,
two 96-well plates for assessment of cloning efficiency (viable clones)
and four 96-well plates for assessment of mutant potential.

NUMBER OF CELLS EVALUATED: 2000 cells/well x 384 wells = 768000 cells per test concentration, negative, vehicle or positive control (per culture)
corresponding to 1536000 cells per test concentration, negative, vehicle or positive control (sum from both cultures).

DETERMINATION OF CYTOTOXICITY: Relative survival corrected with the post treatment cell concentrations;
[in preliminary toxicity test relative survival after treatment (Day 0) and on Days 1 and 2]

Evaluation criteria:

Mutagenicity of the test material was considered to be evident if all of the following criteria were met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle (solvent) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10^6 viable cells
(Global Evaluation Factor, GEF according to Moore et al. 2006) higher than the corresponding vehicle (solvent) control value.

Reference for GEF:
Moore, M.M., Honma, M., Clements, J., Bolcsfoldi, G., Burlinson, B. Cifone, M., Clarke, J., Delongchamp, R., Durward, R., Fellows, M., Gollapudi, B., Hou, S., Jenkinson, P., Lloyd, M., Majeska, J., Myhr, B., O’Donovan, M, Omori, T, Riach, C., San, R., Stankowski. JR. L.F., Thakur, A.K., Van Goethem, F., Wakuri, S. and Yoshimura, I. (2006). Mouse lymphoma thymidine kinase gene mutation assay: Follow-up meeting of the international workshop on Genotoxicity testing – Aberdeen, Scotland, 2003 – Assay acceptance criteria, positive controls, and data evaluation. Environmental and Molecular Mutagenesis. 47, 1-5.

Statistics:

Microsoft Excel 2000 software was used for statistical analysis of mutant frequencies (total wells with clones). Log mutant frequency (LMF) of the vehicle control group was compared with the LMF of each treatment group, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore any negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

Preliminary Toxicity Testing: 3 h exposure (–/+S9) and 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

not applicable

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment 1, 3 h exposure (–/+S9) & Experiment 2, 3 h exposure (+S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiments 2 & 3, 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
In the present study variations in osmolality and pH between vehicle control and test material treated culture media were within acceptable limits, but cytotoxicity of the test material was a confounding factor. Precipitate in test material treated medium was only evident in the preliminary toxicity test, but this did not affect the selection of test concentrations for the subsequent main assays.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation (-/+S9)