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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro Ames test, the in vitro chromosome aberration test in human lymphocytes and the in vitro mutation test with mouse lymphoma L5178Y cells were chosen as key studies for genetic toxicity, as they represent different aspects of genetic toxicity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
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 an internationally accepted technical guideline and in compliance with GLP in a contract research organization.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Deviations:
yes
Remarks:
Occasional incubation temperature from 34.2°C to 39.9°C. In view of good growth and comparable results in all tests, study results were considered to be unaffected by this.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA102, TA1535, TA1537
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver microsomal fraction (S9 mix), supplied by Molecular Toxicology, Inc., Boone, North Carolina, USA
Test concentrations with justification for top dose:
Preliminary toxicity test (only with TA100): 5.0, 12.5, 25, 50, 125, 250, 500, 1250, and 5000 μg/plate
Main Tests (Experiments 1 and 2, with all strains): 0.50, 5.0, 50, 500, and 5000 μg/plate

In addition, in several instances, 0.05 and 0.005 μg/plate levels were also assayed, but being all negative for mutagenicity these results in general were not reported. Only for the independent repeat experiment (Experiment 2) for TA98 (-S9) the results of the 0.05 µg/plate level were reported, because in this experiment no data were available for the 0.5 µg/plate concentration, as no background lawn was observed.
Vehicle / solvent:
Ethanol
Justification for choice of solvent/vehicle: Outcome of solubility trials conducted at the testing laboratory.
Untreated negative controls:
yes
Remarks:
testing for spontaneous reversion and in some assays with TA100, TA102 and TA1535 mainly (-S9) water controls included
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene and mitomycin C.
Details on test system and experimental conditions:
Two independent assays (Plate Incorporation tests) were conducted (Experiments 1 and 2), each without and with metabolic activation (-/+ S9 mix)

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Water and vehicle controls: all plating performed in duplicate; Test concentrations: all plating performed in triplicate.
Experiments 1 and 2: All test substance concentrations, vehicle controls and positive controls were plated in triplicate. On very few occasions single plates were not considered due to reduced background lawn.

PRECIPITATE:
In the main tests (Experiments 1 and 2) moderate precipitate was evident at 5000 µg/plate in all but one assays. Only in Experiment 2 with TA1535 (-S9 mix) precipitate was not recorded at 5000 µg/plate. In the preliminary toxicity test, precipitate was moderate in degree at 2500 µg/plate and moderat to heavy in degree 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: 1.5 μg/plate: - strains: TA 1535, TA 100
9-Aminoacridine: 75 μg/plate: - strain: TA 1537
2-Nitrofluorene: 2.0 μg/plate: - strain: TA 98
Mitomycin C: 0.5 μg/plate: - strain: TA 102

With metabolic activation (+S9 mix):
2-Aminoanthracene: 1.0 μg/plate: - strains: TA 98, TA 100, TA 102, TA 1535, TA 1537

DURATION
Incubation time: 48 h at approximately 37 +/- 2 degrees. Occasional incubation temperature from 34.2°C to 39.9°C. In view of good growth and comparable results in all tests, study results were considered to be unaffected by this.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was tested in the preliminary assay with a single strain, TA100, in the presence and absence of metabolic activation.

TESTER STRAIN GENOTYPE CONFIRMATION
Genetic markers, such as histidine/biotin requirement, crystal violet sensitivity, ampicillin resistance, ultra violet sensitivity, tetracycline resistance and spontaneous reversion rates of the cultures to histidine independence have been checked in the testing laboratory. Each strain demonstrated the strain genotypes expected. In addition cell titres of each strain were measured at use.

Evaluation criteria:
A concentration was considered toxic if one or both of the following criteria were met:
1) a reduction greater than 50% in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction hasd to be accompanied by an abrupt dose-dependent drop on the revertant count.
2) A reduction observed in the background lawn.

The test substance was considered positive in the assay when:
1) statistically significant dose-related increase in the mean number of revertants for at least one tester strain, over a minimum of two test concentrations. Data sets for strains TA98, TA100 and TA102 were considered positive if the mean number of revertants at the peak of the dose-response was two times (or greater) than the mean solvent control values. Data sets for strains TA1535 and TA1537 were considered positive if the mean number of revertants at the peak of the dose-response was three times (or greater) than the mean solvent control values.
Statistics:
Not specified.
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
In all but one incidence, precipitate moderate in degree at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There was no evidence of reproducible cytotoxicity at any of the concentrations tested.

Positive controls were considered to be valid as in general their revertant colony number values were more than threefold higher than those of concurrent vehicle controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)
Conclusions:
Interpretation of results (migrated information):
negative without and with metabolic activation (S9 mix)

In both main experiments, the test material was found to be non-mutagenic for all of the tested Salmonella strains, TA98, TA100, TA102, TA1535 and TA1537, both without and with metabolic activation.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
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.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
of 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
of 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
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): 0*, 3.92, 6.53, 10.88, 18.14, 30.23, 50.39, 83.98, 139.97, 233.28, 388.88, 648 and 1080 µg/mL.
Because of inappropriate toxicity in the above test without and with S9 mix, a repeat test was conducted:
Concentrations prepared without S9 (repeat test): 0*, 80, 120, 160, 200, 240, 280, 320, 360, 400 and 440 μg/mL.
Concentrations prepared with S9 (repeat test): 0*, 200, 400, 525, 650, 775, 900, 1025, 1150, 1275, 1400, 1600 and 1800 μg/mL.
Microscopically examined (metaphase analysis) without S9: 0*, 160, 240 and 400 μg/mL
Microscopically examined (metaphase analysis) with S9 (2% v/v): 0*, 1275, 1400 and 1800 μg/mL

EXPERIMENT 2:
Concentrations prepared without S9: 0*, 25, 50, 100, 150, 200, 240, 280, 320, 360, 400 and 450 μg/mL.
Concentrations prepared with S9: 0*, 400, 800, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 and 1800 μg/mL.
Because of variable toxicity (of no biological relevance) across the concentration range without S9 mix, a repeat test was conducted to define an acceptable concentration range:
Concentrations prepared without S9 (repeat test): 0*, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 and 275 g/mL.
Microscopically examined (metaphase analysis) without S9: 0*, 25, 75 and 200 µg/mL
Microscopically examined (metaphase analysis) with S9 (5% v/v): 0*, 1500, 1600 and 1800 µg/mL

*0 μg/mL = vehicle control (ethanol)

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
see the 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 WS400130. WS400130 was shown to be soluble in ethanol at 500 mg/mL. This concentration produced the limit test substance concentration in final culture medium of 5000 µg/mL when administering this ethanolic test substance solution to the culture medium at 1% v/v. Because of visible precipitate at 5000 µg/mL and at lower concentrations, a maximum concentration of 1800 µg/mL was selected for testing in the presence of S9. In the absence of S9, the maximum concentration finally selected for testing was lower than 1800 µg/mL due to cytotoxicity.


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:
without
Genotoxicity:
negative
Remarks:
In Experiments 1 & 2, following 3 h and 21 h treatment, respectively.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In each experiment (-S9) the relative mitotic index was 50% at the highest concentration selected for metaphase analysis.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Following 3 h treatment in Experiments 1 & 2, with 2% & 5% S9 fraction in final culture medium, respectively.
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
in both experiments.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
In the absence of S9, cytotoxicity was a confounding factor in both experiments (Experiments 1 & 2), leading to the selection of 400 µg/mL after 3 h exposure and 200 µg/mL after 21 h exposure as maximum concentration for metaphase analysis.
In the presence of S9, visible precipitate was a confounding factor in both experiments, leading to the selection of the highest non-precipitating concentration and two precipitating concentrations for metaphase analysis in each experiment. The highest precipitating concentration tested in each experiment in the presence of S9 was 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: strain/cell type: human lymphocytes with 44- 48 chromosomes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative Without and with metabolic activation (-/+S9)
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
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.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
of 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
of 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 media, 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 media, 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):
9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 μg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 5, 10, 25, 50, 75, 100, 200, 300, 400, 500 and 600 μg/mL
Mutant phenotype determination at: 25, 50, 100, 200, 500 and 600 μg/mL

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 10, 100, 200, 400, 500, 600, 700, 800, 900 and 1000 μg/mL
Mutant phenotype determination at: 10, 100, 200, 400, 800 and 900 μg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 5, 25, 50, 60, 70, 80, 90 and 100 μg/mL
Mutant phenotype determination at: 5, 25, 50, 60 and 70 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 WS400130. WS400130 was shown to be soluble in ethanol at 500 mg/mL. This concentration produced a 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 this concentration, the pH of the medium was acceptably similar to that of vehicle control medium. However, at 5000 µg/mL fluctuation in osmolality was > 50 mOsm/kg compared with the vehicle control. At 2500 µg/mL fluctuation in osmolality was within accepptable limits leading to the choice of 2500 µg/mL as the maximum concentration tested in the preliminary toxicity test.
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
Remarks:
Positive control substance for tests without metabolic activation (-S9) in Experiments 1 and 2 Migrated to IUCLID6: 3h exposure: 10 µg/mL; 24 h exposure: 5 µg/mL, vehicle DMSO
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 5000 µg/mL fluctuation in osmolality was unacceptably high (i.e. > 50 mOsm/kg compared with the vehicle control) leading to the choice of 2500 µg/mL as the maximum concentration tested in the preliminary toxicity test. The concentrations chosen for mutagenicity testing In both main mutation experiments (Experiments 1 and 2) were considerably lower than 2500 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. In addition, 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).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 

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

WS400130
(9.8)

82

WS400130
(9.8)

98

WS400130
(9.8)

91

WS400130
(19.5)

65

WS400130
(19.5)

111

WS400130
(19.5)

71

WS400130
(39.1)

49

WS400130
(39.1)

93

WS400130
(39.1)

52

WS400130
(78.1)

49

WS400130
(78.1)

86

WS400130
(78.1)

16

WS400130
(156.3)

48

WS400130
(156.3)

69

WS400130
(156.3)

2

WS400130
(312.5)

32

WS400130
(312.5)

43

WS400130
(312.5)

0

WS400130
     (625)(p)

 3

WS400130
     (625)(p)

12

WS400130
(625)

0

WS400130
   (1250)(p)

11

WS400130
   (1250)(p)

5

WS400130
   (1250)(p)

0

WS400130
   (2500)(p)

22

WS400130
   (2500)(p)

18

WS400130
   (2500)(p)

0

 

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

 

Table 2:

Precipitation and 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

 

 

 

 

WS400130
(5)

79

 

 

WS400130
(10)

102

 

 

WS400130
(25)

80

 

 

WS400130
(25)

71

WS400130
(50)

55

 

 

WS400130
(50)

57

 

 

 

 

WS400130
(60)

24

 

 

 

 

WS400130
(70)

15

WS400130
(100)

46

WS400130
(100)

68

 

 

WS400130
(200)

27

WS400130
(200)

49

 

 

 

 

WS400130
(400)

32

 

 

WS400130
(500)

21

 

 

 

 

WS400130
(600)

17

 

 

 

 

 

 

WS400130
(800)

22

 

 

 

 

WS400130
(900)

16

 

 

 

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

Conclusions:
Interpretation of results (migrated information):
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

Additional information

Justification for classification or non-classification

Based on the negative results attained in all in vitro genotoxicity studies WS400130 is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].