Fatty acids, C18-unsatd., trimers, reaction products with triethylenetetramine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

Experiment 1 [each strain was tested without and with metabolic activation (-/+S9, respectively)]
Doses: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate

Experiment 2:
TA 98 (-/+S9): 0; 15; 50; 150; 500; 1500 μg/plate
TA 100 (-/+S9): 0; 50; 150; 500; 1500; 5000 μg/plate
TA 1535 (-/+S9): 0; 15; 50; 150; 500; 1500 μg/plate
TA 1537 (- S9): 0; 5; 15; 50; 150; 500 μg/plate
TA 1537 (+S9): 0; 15; 50; 150; 500; 1500 μg/plate
WP2uvrA (-/+S9): 0; 50; 150; 500; 1500; 5000 μg/plate

Vehicle / solvent:

Ethanol
Justification for choice of solvent/vehicle:
Ethanol was a suitable vehicle for exposure to the test substance up to the maximum guideline recommended test substance concentration of of 5000 μg/plate.

Untreated negative controls:

other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide; 9-aminoacridine; 2-Nitrofluorene; 4-nitroquinoline-1-oxide.

Remarks:

Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.

Untreated negative controls:

other: Sterility controls were included, i.e. tester strain free plates with soft agar, S9 mix, buffer, vehicle and/or test substance.

Negative solvent / vehicle controls:

yes

Remarks:

without and with S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; Benzo[a]pyrene

Remarks:

Positive control substances for tests with metabolic activation (S9 mix). All of them are well established reference mutagens.

Details on test system and experimental conditions:

Standard Plate Incorporation Tests were performed in both experiments (Experiments 1 and 2) and both experiments were conducted without and with metabolic activation (S9 mix). Test procedures varied in that the proportion of S9 fraction in the S9 mix was 10% in Experiment 1 whereas 20% in Experiment 2.

In both experiments, precipitate was observed on all plates containing WS400152 at 5000 μg/plate.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (S9 mix):

Sodium azide (CAS No. 26628-22-8):
- 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100

9-Aminoacridine (CAS No. 90-45-9):
- 50 μg/plate, dissolved in DMSO: - strain: TA 1537

2-Nitrofluorene (CAS No. 607-57-8):
- 2 μg/plate, dissolved in DMSO: - strain: TA 98

4-Nitroquinoline-1-oxide (CAS No. 56-57-5):
- 2 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

With metabolic activation (S9 mix):

2-Aminoanthracene (CAS No. 613-13-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 100
- 10 μg/plate, dissolved in DMSO: - strain: WP2 uvrA

Benzo[a]pyrene (CAS No. 50-32-8):
- 5 μg/plate, dissolved in DMSO: - strains: TA 1537, TA 98

Evaluation criteria:

The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
A reproducible increase in revertant colony number, (i.e. at least twice for strains TA 100, TA 98 and WP2 uvrA and at least three times for strains TA 1535 and TA 1537 the concurrent vehicle controls), with some evidence of a positive dose-response relationship. Such positive response in at least one tester strain without or with metabolic activation (S9 mix.) is sufficient for concluding mutagenic activity.

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

Statistics:

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments (Experiment 1 and 2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

lowest concentration at which toxicity was observed = 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

lowest concentration at which toxicity was observed = 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

lowest concentration at which toxicity was observed = 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

All sterility control plates were colony free. Hence the absence of microbial contamination of the S9 mix, buffer and test substance formulation was confirmed. Viability counts were satisfactory meeting the acceptance criteria.

Conclusions:

Interpretation of results: negative without and with metabolic activation (S9 mix)

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

of 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Species / strain / cell type:

lymphocytes: human, cultured in vitro in whole blood culture

Details on mammalian cell type (if applicable):

- Source of lymphocytes: Human blood collected aseptically from two healthy, non-smoking male donors and pooled.
- Type and identity of media:
RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Sprague Dawley derived rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

EXPERIMENT 1:
Concentrations prepared without and with metabolic activation (-/+S9, respectively) : 0*, 18.14, 30.23, 50.39, 83.98, 139.97, 233.28, 388.80, 648, 1080, 1800 µg/mL.
Because of inappropriate toxicity in the above test in the presence of S9 mix, repeat tests were conducted to define an acceptable concentration range:
Concentrations prepared +S9 (repeat test): 0*, 2.5, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 90, 110, 130, 150 μg/mL
Concentrations prepared +S9 (further repeat test): 0*, 2.5, 5, 10, 50, 100, 130, 135, 140, 145, 150 μg/mL
Microscopically examined (metaphase analysis) -S9: 0*, 18.14, 30.23, 50.39 μg/mL
Microscopically examined (metaphase analysis) +S9 (2% v/v): 0*, 10, 100, 140 μg/mL

EXPERIMENT 2:
Concentrations prepared -S9: 0*, 2.5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg/mL
Concentrations prepared +S9: 0*, 5, 50, 100, 110, 120, 130, 135, 140, 145, 150 μg/mL
Because of inappropriate toxicity in the above test in the presence of S9 mix, repeat tests were conducted to define an acceptable concentration range:
Concentrations prepared +S9 (repeat test): 0*, 2.93, 4.19, 5.98, 8.55, 12.21, 17.40, 24.91, 33.60, 50.85, 72.64, 103.77, 148.24, 211.77, 302.53, 432.18, 617.40, 882, 1260, 1800 µg/mL
Concentrations prepared +S9 (further repeat test): 0*, 2.5, 5, 10, 50, 100, 150, 200, 210, 225, 240, 255, 270, 285, 300 μg/mL
Microscopically examined (metaphase analysis) -S9: 0*, 2.5, 45, 50 µg/mL
Microscopically examined (metaphase analysis) +S9 (5% v/v): 0*, 10, 100, 210 µg/mL

*0 μg/mL = vehicle control (ethanol)

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
see field "Any other information on materials and methods incl. tables"

Vehicle / solvent:

Ethanol

Justification for choice of solvent/vehicle:
Ethanol was chosen as a vehicle to maximise exposure of cultures in the test system to WS400152. WS400152 was shown to be soluble in ethanol at 500 mg/mL. This concentration produced the limit test substance concentration in culture medium of 5000 µg/mL when administering this ethanolic test substance solution to the culture medium at 1% v/v. At 3000 and 5000 µg/mL, osmolality of the medium was acceptably similar to that of vehicle control medium (i.e. fluctuation ≤ 50 mOsm/kg). However, at both concentrations fluctuation in pH of more than 1.0 unit was observed compared with the vehicle control leading to the choice of 1800 µg/mL as the maximum concentration tested in the present study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C tested at 0.2 μg/mL (3 hour treatment) and 0.1 μg/mL (21 hour continuous treatment), vehicle sterile purified water Migrated to IUCLID6: without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide tested at 5 μg/mL (3 hour treatment), vehicle sterile purified water Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

CELL DIVISION STIMULANT:
Phytohaemagglutinin

METHOD OF APPLICATION:
in cell culture medium;

DURATION
- Exposure duration:
3 hours in Experiment 1 (without and with metabolic activation) and in Experiment 2 (with metabolic activation).
21 hours in Experiment 2 (without metabolic activation)
[After the 3 h treatment the cells were cultivated with fresh media for 18 h].
- Concentration of S9 fraction in final medium:
Experiment 1: 2 % v/v
Experiment 2: 5 % v/v
- Fixation time (start of exposure up to fixation or harvest of cells):
21 hours in each of both experiments.

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® was added to the cultures (0.1 µg/mL culture medium) 19 hours after treatment start.
2 h later, the cells were treated with hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.

STAIN (for cytogenetic assays):
After fixation the cells were stained with 10% Giemsa.

NUMBER OF REPLICATIONS:
Duplicate cultures were treated at each concentration.

NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.
This number was reduced in cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) determined by counting the number of mitotic cells in 1000 cells;

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (including and excluding gaps),
- Number of gaps,
- Types of aberrations
Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap,
Others: Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes

Determination of polyploidy:
- Polyploid and endoreduplicated cells were noted when seen.

Evaluation criteria:

An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
-Significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) at one or more test concentration.
-The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.

Statistics:

One-tailed Fisher exact test (Fisher 1973) for comparison of the number of aberrant metaphase cells in each test substance group with the vehicle control value.

In addition, a Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

ARMITAGE, P. (1955) Tests for linear trends in proportions and frequencies. Biometrics, 11, 375-386. (Cochran-Armitage test).
FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see Test concentrations under Methods

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At concentrations of 3000 and 5000 µg/mL fluctuation in pH of more than 1.0 unit was observed compared with the vehicle control leading to the choice of 1800 µg/mL as the maximum concentration tested. Due to cytotoxicity the concentrations selected for metaphase analysis were considerably lower than 1800 µg/mL.

ADDITIONAL OBSERVATIONS DURING METAPHASE ANALYSIS
Statistically significant increases in polyploid metaphases or notable increases in endoreduplicated metaphases were not evident.

Remarks on result:

other: no genotoxicity observed

Conclusions:

Interpretation of results : negative Without and with metabolic activation (-/+S9)

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

of 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of medium, in general used for cell culture:
R10p, i.e. medium R0 supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v,
whereby medium R0 is RPMI 1640 buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.

- Type and identity of medium, used for cloning efficiency plating:
R20p prepared by mixing equal volumes of R10p and R30p,
whereby R30p is medium R0 supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.

HiDHS = heat-inactivated donor horse serum

- 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 Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone for enzyme induction.

Test concentrations with justification for top dose:

PRELIMINARY TOXICITY TESTING (suspension growth 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):
3.5, 7, 14.1, 28.1, 56.3, 112.5, 225, 450, 900 and 1800 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 10, 20, 30, 40, 50, 60, 80 and 100 µg/mL
Mutant phenotype determination at: 10, 20, 30 and 40 µg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 10, 25, 50, 75, 100, 125, 175 and 200 µg/mL
Mutant phenotype determination at: 10, 25, 50, 75, 100 and 125 µg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 5, 10, 20, 30, 40, 50, 60, 80 and 100 µg/mL
Mutant phenotype determination at: 5, 10, 20, 30 and 40 µg/mL

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR MUTANT PHENOTYPE DETERMINATION:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (i.e. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% relative total growth (RTG).

Vehicle / solvent:

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

Justification for choice of solvent/vehicle:
Ethanol was chosen as a vehicle to maximise exposure of cultures in the test system to WS400152. WS400152 was shown to be soluble in ethanol at 500 mg/mL. This concentration produced the limit test substance concentration in culture medium of 5000 µg/mL when administering this ethanolic test substance solution to the culture medium at 1% v/v. Fluctuation in osmolality was within acceptable limits at final test substance concentrations in culture medium of 1800 and 5000 µg/mL. However, changes in pH of the medium were unacceptably high (i.e. > 1 unit compared with the vehicle control) at greater 1800 µg/mL and precipitate was evident at 3000 µg/mL and greater concentrations. In view of the changes in pH, the maximum concentration tested in the preliminary toxicity test was 1800 µg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

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

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

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

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/mL, vehicle DMSO

Positive control substance:

benzo(a)pyrene

Remarks:

Positive control substance for tests with metabolic activation (+S9) in Experiment 1

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: 24 h exposure without metabolic activation (–S9)

- Selection time: At 48 h after the end of exposure addition of the selection agent trifluorothymidine (TFT)
then allowing 10-14 days for cells to grow with TFT.

SELECTION AGENT: Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 cultures at each concentration,
[from each culture two vials for assessment of growth in suspension, two 96-well plates for assessment of cloning efficiency
and two 96-well plates for assessment of mutant potential; vehicle controls in quadruplicate].

NUMBER OF CELLS EVALUATED: 2000 cells/well x 192 wells = 384000 cells per culture

DETERMINATION OF CYTOTOXICITY: Relative total growth; (in preliminary toxicity test Relative suspension growth)

Evaluation criteria:

The mutation test result was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF = 126 x 10^–6, Moore et al. 2006, detailed reference see below).

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied: If the linear trend test was negative, the result was regarded as negative. If the linear trend test was positive, this indicated a positive, biologically relevant response.

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:

The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. 1989 using a one-sided F-test, where p<0.001.
Robinson, W.D., Green, M.H.L., Cole, J., Healy, M.J.R., Garner, R.C., and Gatehouse, D. (1989). Statistical evaluation of bacterial/mammalian fluctuation tests. In: Kirkland, D. J. (Ed). UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part 111. Statistical Evaluation of Mutagenicity Test Data, p.102-140. Cambridge University Press, Cambridge.

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

Remarks:

Detailed in Table 1

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment 1, 3 h exposure (–/+S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Detailed in Table 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment 2, 24 h exposure (–S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Detailed in Table 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
At concentrations > 1800 µg/mL changes in pH of the medium were unacceptably high (i.e. > 1 unit compared with the vehicle control) leading to the choice of 1800 µg/mL as the maximum concentration in the preliminary toxicity test. The concentrations chosen for mutagenicity testing In both main mutation experiments (Experiments 1 and 2) were considerably lower than 1800 µg/mL because of cytotoxicity (detailed in Table 2).

JUSTIFICATION FOR NOT CONFIRMING THE NEGATIVE RESULT ATTAINED WITH METABOLIC ACTIVATION (+S9)
In the presence of S9, there were no increases in mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical solvent control values and there were no increases in mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore, it was considered unnecessary to perform a direct repeat of the assay in the presence of S9. In the absence of S9, the negative mutagenicity results attained after 24 h of exposure (Experiment 2) confirmed those attained after 3 hours of exposure (Experiment 1).

 

Table 1: Precipitation and Cytotoxicity Expressed as Relative Suspension Growth (RSG) in the Preliminary Toxicity Test
3 h exposure (–S9)		3 h exposure (+S9)		24 h exposure (–S9)
Treatment / Concentration (µg/mL)	RSG (%)	Treatment / Concentration (µg/mL)	RSG (%)	Treatment / Concentration (µg/mL)	RSG (%)
Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100
WS400152 (3.5)	131	WS400152 (3.5)	74	WS400152 (3.5)	102
WS400152 (7)	129	WS400152 (7)	103	WS400152 (7)	102
WS400152 (14.1)	84	WS400152 (14.1)	107	WS400152 (14.1)	84
WS400152 (28.1)	62	WS400152 (28.1)	93	WS400152 (28.1)	71
WS400152 (56.3)	5	WS400152 (56.3)	72	WS400152 (56.3)	15
WS400152 (112.5)	3	WS400152 (112.5)	30	WS400152 (112.5)	1
WS400152 (225)	4	WS400152 (225)	5	WS400152 (225)	1
WS400152 (450)	6	WS400152 (450)	6	WS400152    (450)(p)	1
WS400152    (900)(p)	4	WS400152    (900)(p)	3	WS400152    (900)(p)	1
WS400152   (1800)(p)	1	WS400152   (1800)(p)	0	WS400152   (1800)(p)	1

 

(p):  Precipitate observed by eye at the end of treatment  

Table 2: Cytotoxicity Expressed as Mean Relative Total Growth (RTG) in Both Main Mutation Experiments
Experiment 1, 3 h exposure (–S9)		Experiment 1, 3 h exposure (+S9)		Experiment 2, 24 h exposure (–S9)
Treatment / Concentration (µg/mL)	RTG (%)	Treatment / Concentration (µg/mL)	RTG (%)	Treatment / Concentration (µg/mL)	RTG (%)
Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100	Vehicle Control (Ethanol)	100
				WS400152 (5)	101
WS400152 (10)	77	WS400152 (10)	122	WS400152 (10)	110
WS400152 (20)	65			WS400152 (20)	74
		WS400152 (25)	102
WS400152 (30)	28			WS400152 (30)	43
WS400152 (40)	10			WS400152 (40)	4
		WS400152 (50)	89
		WS400152 (75)	33
		WS400152 (100)	15
		WS400152 (125)	11

Precipitation was not seen at any exposure concentrations in Experiments 1 and 2.

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative results attained in all in vitro genotoxicity studies WS400152 is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].