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Genetic toxicity in vitro

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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:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix (supplied by MOL. TOX Molecular Toxicology).
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: essential amino acid requiring strain
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix (supplied by MOL. TOX Molecular Toxicology).
Test concentrations with justification for top dose:
Preliminary screening test (only with TA100 & WP2 uvrA): 5.0, 1.0, 0.5, 0.1 and 0.05 mg/plate
Main Tests (Experiments 1 and 2, with all strains): 5.0, 1.0, 0.5, 0.1 and 0.05 mg/plate
Vehicle:
DMSO
Negative controls:
yes
Remarks:
testing for spontaneous reversion
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 2-nitrofluorene, 2-aminoanthracene and 9-aminoacridine
Remarks:
for Salmonella strains
Negative controls:
yes
Remarks:
testing for spontaneous reversion
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene and methyl methanesulfonate
Remarks:
for Escherichia coli
Details on test system and 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: All plating performed in triplicate

Evaluation of toxicity was based on reversion frequency, viability and integrity of the background lawn.

Precipitate was not evident at any tested concentration of WS400517.

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: 5 μg/plate: - strains: TA 1535, TA 100
9-Aminoacridine: 100 μg/plate: - strain: TA 1537
2-Nitrofluorene: 5 μg/plate: - strain: TA 98
Methyl methanesulfonate: 0.001 mL/plate: - strain: WP2 uvrA

With metabolic activation (+S9 mix):
2-Aminoanthracene: 1.0 μg/plate: - strains: TA 98, TA 100, TA 1535, TA 1537
10 μg/plate: - strain: WP2 uvrA

Genetic markers, such as histidine/biotin requirement, crystal violet sensitivity, ampicillin resistance, ultra violet sensitivity, tetracycline resistance for Salmonella strains and tryptophan requirement for growth and sensitivity to Mitomycin C for E. coli, and spontaneous reversion rates have been routinely checked in the testing laboratory.
Evaluation criteria:
The test material was considered to exhibit mutagenic activity in this assay if the following criteria were met:
1) there was a statistically significant difference (at p < = 0.05) in the number of revertants between the solvent control and the test material, as determined by One Way Analysis of Variance (Anova).
2) In response to a test material concentration, strain TA98 or TA100 responded with a mean reversion frequency twofold or more greater than that of the corresponding solvent control plates, or strains TA1535, TA1537 or WP2 uvrA responded with a mean reversion frequency threefold or more greater than that of the corresponding solvent control plates. In addition, the response must be dose-dependent or increasing concentrations of the test material must show increasing mean reversion frequencies. In evaluating the results, the magnitude of any increase in reversion frequency and the degree of associated toxicity (if any) is given due consideration.

Accordingly, the response of a test material was considered to be negative if (1) the Anova did not disclose a statistically significant difference (at p = 0.05) from the corresponding solvent control, (2) the increases (if any) in mean reversion frequency for TA98 or TA100 were < twofold and that (if any) for TA1535, TA1537 or WP2 uvrA were < threefold that of the corresponding solvent control plates and (3) there was no evidence of a dose-dependent response.

A response was considered equivocal if it did not fulfil the criteria of either a negative or a positive response and/or the Study Director did not consider the response to be either positive or negative.
Statistics:
Numbers of revertant conlonies were statistically analysed using One Way Analysis of Variance to determine statistically significant differences of test concentrations or positive controls from vehicle controls at p<=0.05.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
of 5000 µg/plate, without precipitate
Vehicle controls valid:
other: See "Additional information on results"
Negative controls valid:
not applicable
Positive controls valid:
other: See "Additional information on results"
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
of 5000 µg/plate, without precipitate
Vehicle controls valid:
other: See "Additional information on results"
Negative controls valid:
not applicable
Positive controls valid:
other: See "Additional information on results"
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)
Additional information on results:
The historical reference ranges for vehicle and positive controls were not presented in the study report and therefore the validity of the controls could not be assessed. However, the presented revertant colony number values of concurrent negative controls were similarly low to those of the test material treated groups, whilst those of positive controls were substantially higher (statistically significant, p<0.05), consistent with what would be expected from valid negative and positive controls. In addition, the numbers of spontaneous revertants/plate were similarly low to those of the corresponding vehicle controls.
Conclusions:
negative without and with metabolic activation (S9 mix)

In both main experiments, the test material was found to be non-mutagenic and non-cytotoxic at 5000 µg/plate and below this level for all of the tested Salmonella and E. coli strains, both with and without 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:
guideline study
Reference:
Composition 0
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
Test material information:
Composition 1
Species / strain:
lymphocytes: human, cultured in vitro in whole blood culture
Details on mammalian cell lines (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 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*, 29.94, 49.90, 83.17, 138.61, 231.02, 385.04, 641.74, 1069.56, 1782.60, 2971 µg/mL
Microscopically examined (metaphase analysis) without S9: 0*, 385.04, 641.74 and 1069.56 μg/mL
Microscopically examined (metaphase analysis) with S9 (2% v/v): 0*, 641.74, 1069.56 and 2971 μg/mL

EXPERIMENT 2:
Concentrations prepared without and with metabolic activation (S9): 0*, 83.17, 138.61, 231.02, 385.04, 641.74, 1069.56, 1782.60, 2971 µg/mL
Microscopically examined (metaphase analysis) without S9: 0*, 231.02, 385.04 and 641.74 µg/mL
Microscopically examined (metaphase analysis) with S9 (5% v/v): 0*, 231.02, 385.04 and 641.74 µg/mL

*0 μg/mL = vehicle control (dimethyl sulphoxide, DMSO)

CRITERIA FOR SELECTING APPROPRIATE TEST CONCENTRATIONS FOR METAPHASE ANALYSIS:
In the present study, cytotoxicity was not a limiting factor in the selection of test concentrations for metaphase analysis, as the relative mitotic index was well above 50% in all tests. Justification for concentration selection was based on visible precipitate in the final culture medium at the end of treatment. In general (except Experiment 1 with metabolic activation), the highest concentration selected for analysis was the lowest concentration causing visible precipitate in the final culture medium at the end of treatment. In Experiment 1 (+S9) two concentrations with visible precipitate and one lower concentration were selected for metaphase analysis.
Vehicle:
Dimethyl sulphoxide (DMSO)

Justification for choice of solvent/vehicle:
DMSO was chosen as a vehicle to maximise exposure of cultures in the test system to WS400517. WS400517 was shown to be soluble in DMSO at 297.1 mg/mL representing the limit of solubility in a compatible vehicle. This concentration produced a test substance concentration in final culture medium of 2971 µg/mL when administering this DMSO test substance solution to the culture medium at 1% v/v. Fluctuation in osmolality and pH were within acceptable limits at final test substance concentrations in culture medium of 2971 µg/mL. In this case, the highest final concentration used for subsequent testing was 2971 µg/mL as this maintained visible precipitate in culture medium in at least two concentrations.


Negative controls:
no
Solvent 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
Negative controls:
no
Solvent 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 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 both experiments, following 3 h or 21 h continuous treatment
Cytotoxicity:
no, but tested up to precipitating concentrations
Remarks:
In both experiments, following 3 h or 21 h continuous treatment
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: human lymphocytes with 44- 48 chromosomes
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
A statistically significant increase in chromosome aberrations (p<0.01; excluding gaps only) at the highest concentration (Table 1, Experiment 1, at 2971 µg/mL) was not considered to be biologically relevant.
Cytotoxicity:
no, but tested up to precipitating concentrations
Remarks:
In both experiments following 3 h treatment
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: human lymphocytes with 44- 48 chromosomes
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
In Experiment 1, precipitate was present at WS400517 concentrations of 1069.56 μg/mL and above in final culture medium and in Experiment 2 precipitate was evident at final concentrations of 641.74 μg/mL and above, both without and with metabolic activation.

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

Table 1:    Experiment 1

Chromosome Aberration and Cytotoxicity Results Based on Duplicate Cultures/Concentration

 

Exposure period

S9 mix

Nominal concentration of WS400517

Cells with aberrations excluding gaps

Cells with aberrations including gaps

Relative

mitotic

index

(%)

Polyploidy

mean

incidence

(%)

(hours)

(v/v)

(µg/mL)

Individual values (%)

Mean (%)

Individual values (%)

Mean (%)

 3

-

0 (DMSO)

 3.0

 0.0

 1.5

 4.0

 2.0

 3.0

100

0.0

 

 

385.04

 2.0

 2.0

 2.0

 2.0

 4.0

 3.0

101

0.5

 

 

641.74

 0.0

 3.0

 1.5

 0.0

 4.0

 2.0

102

0.0

 

 

1069.56PPT

 0.0

 0.0

 0.0

 0.0

 1.0

 0.5

108

0.0

 

 

0.2 (Mitomycin C)

 25.6

 27.8

 26.7***

 25.6

 30.6

 28.0***

-

0.0

 

 

 

 

 

 

 

 

 

 

 

 3

+

0 (DMSO)

 0.0

 0.0

 0.0

 0.0

 2.0

 1.0

100

1.0

 

(2%)

641.74

 1.0

 0.0

 0.5

 1.0

 0.0

 0.5

106

0.0

 

 

1069.56PPT

 2.0

 0.0

 1.0

 2.0

 1.0

 1.5

96

0.5

 

 

2971PPT

 2.0

 5.0

 3.5**

 3.0

 5.0

 4.0

71

0.5

 

 

5 (Cyclophosphamide)

 32.3

 37.0

 34.5***

 35.5

 40.7

 37.9***

-

1.7

 

One-tailed Fisher's exact test

***                    p<0.001

**                      p<0.01

Otherwise          p>0.01

PPT                                    Visible precipitate present in the final culture medium at the end of treatment.

 

 

Table 2:    Experiment 2

Chromosome Aberration and Cytotoxicity Results Based on Duplicate Cultures/Concentration

 

Exposure period

S9 mix

Nominal concentration of WS400517

Cells with aberrations excluding gaps

Cells with aberrations including gaps

Relative

mitotic

index

(%)

Polyploidy

mean

incidence

(%)

 

(hours)

(v/v)

(µg/mL)

Individual values (%)

Mean (%)

Individual values (%)

Mean (%)

 

21

-

0 (DMSO)

 0.0

 0.0

 0.0

 0.0

 0.0

 0.0

100

0.5

 

 

 

231.02

 1.0

 1.0

 1.0

 1.0

 2.0

 1.5

106

0.0

 

 

 

385.04

 1.0

 1.0

 1.0

 1.0

 3.0

 2.0

95

0.0

 

 

 

   641.74PPT

 1.0

 1.0

 1.0

 4.0

 2.0

 3.0

97

0.0

 

 

 

0.1 (Mitomycin C)

 27.0

 23.3

 25.0***

 29.7

 27.9

 28.8***

-

0.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 3

+

0 (DMSO)

 0.0

 0.0

 0.0

 1.0

 2.0

 1.5

100

0.0

 

 

(5%)

231.02

 1.0

 0.0

 0.5

 1.0

 2.0

 1.5

111

0.0

 

 

 

385.04

 2.0

 2.0

 2.0

 3.0

 2.0

 2.5

104

1.0

 

 

 

   641.74PPT

 1.0

 1.0

 1.0

 2.0

 1.0

 1.5

116

0.0

 

 

 

5 (Cyclophosphamide)

 31.3

 17.9

 22.7***

 37.5

 17.9

 25.0***

-

0.0

 

 

One-tailed Fisher's exact test

***                    p<0.001

Otherwise            p>0.01

PPT                Visible precipitate present in the final culture medium at the end of treatment.

Conclusions:
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:
guideline study
Reference:
Composition 0
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
Test material information:
Composition 1
Target gene:
TK
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (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):
6, 12, 23, 46, 93, 186, 371, 743, 1486 and 2971 μg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (-/+S9):
Exposure concentrations: 93, 186, 371, 743, 1486 and 2971 μg/mL
Mutant phenotype determination at: 93, 186, 371, 743, 1486 and 2971 μg/mL

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 11.63, 46.5, 186, 371, 743, 1486, 2228.3 and 2971 μg/mL
Mutant phenotype determination at: 11.63, 46.5, 186, 371 and 743 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:
Dimethyl sulphoxide (DMSO, 1% v/v final concentration in the medium)

Justification for choice of solvent/vehicle:
DMSO was chosen as a vehicle to maximise exposure of cultures in the test system to WS400517. WS400517 was shown to be soluble in DMSO at 297.1 mg/mL representing the limit of solubility in a compatible vehicle. This concentration produced a test substance concentration in final culture medium of 2971 µg/mL when administering this DMSO test substance solution to the culture medium at 1% v/v. Fluctuation in osmolality and pH were within acceptable limits at final test substance concentrations in culture medium of 138.6 and 2971 µg/mL. A solution of 138.6 mg/mL, dosed at 1% v/v in medium, showed no precipitate in the culture medium, higher concentrations showed precipitate.
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO (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
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO (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 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.
Key result
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:
other: Borderline toxicity, detailed in Table 1
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Experiment 1, 3 h exposure (–/+S9)
Cytotoxicity:
no, but tested up to precipitating concentrations
Remarks:
Detailed in Table 2
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Experiment 2, 24 h exposure (–S9)
Cytotoxicity:
no, but tested up to precipitating concentrations
Remarks:
Detailed in Table 2
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
In view of the limit of solubility of WS400517 in DMSO, the maximum concentration tested in the preliminary toxicity test and and main mutation experiment 1 (3 hour treatment period, -/+ S9) was 2971 µg/mL. The maximum concentration chosen for mutagenicity testing in main mutation experiment 2 was only 743 µg/mL, because of precipitate interfering with scoring at concentrations > 743 µg/mL (detailed in Tables 1 and 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/acceptable 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 (DMSO)

100

Vehicle Control (DMSO)

100

Vehicle Control (DMSO)

100

WS400517
(6)

113

WS400517
(6)

110

WS400517
(6)

90

WS400517
(12)

125

WS400517
(12)

98

WS400517
(12)

98

WS400517
(23)

79

WS400517
(23)

102

WS400517
(23)

84

WS400517
(46)

54

WS400517
(46)

133

WS400517
(46)

87

WS400517
(93)

121

WS400517
(93)

90

WS400517
(93)

85

WS400517
   (186)(p)

91

WS400517
   (186)(p)

74

WS400517
(186)

84

WS400517
   (371)(p)

57

WS400517
   (371)(p)

101

WS400517
     (371)(p)

73

WS400517
   (743)(p)

117

WS400517
   (743)(p)

102

WS400517
   (743)(p)

68

WS400517
 (1486)(p)

92

WS400517
 (1486)(p)

90

WS400517
   (1486)(p)

44

WS400517
 (2971)(p)

105

WS400517
 (2971)(p)

96

WS400517
   (2971)(p)

48

 

(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 (DMSO)

100

Vehicle Control (DMSO)

100

Vehicle Control (DMSO)

100

 

 

 

 

WS400517
(11.63)

147

 

 

 

 

WS400517
(46.5)

101

WS400517
(93)

 97

WS400517
 (93)

 87

 

 

WS400517
   (186)(p)

 72

WS400517
(186)

 86

WS400517
 (186)(p)

102

WS400517
   (371)(p)

 68

WS400517
     (371)(p)

 87

WS400517
 (371)(p)

101

WS400517
   (743)(p)

 71

WS400517
     (743)(p)

 92

WS400517
 (743)(p)

86

WS400517
 (1486)(p)

 94

WS400517
   (1486)(p)

 86

 

 

WS400517
 (2971)(p)

115

WS400517
   (2971)(p )

104

 

 

 

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

 

Conclusions:
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

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.


Short description of key information:
IN VITRO AMES TEST: (according to OECD 471 of 1997):
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A, all negative without and with metabolic activation.

IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST: (according to OECD 473 of 1997):
3 or 21 hours exposure without metabolic activation: all tested concentrations negative
3 hours exposure with metabolic activation (at 2% or 5% S9 fraction in final medium): At the highest tested concentration of WS400517 (2971 µg/mL) statistically significant increase in chromosome aberrations (p< 0.01; excluding gaps only). However, this single finding was not considered to be biologically relevant. All other test concentrations and assays with metabolic activation were negative.

IN VITRO MAMMALIAN CELL GENE MUTATION TEST: (according to OECD 476 of 1997):
3 hours exposure without and with metabolic activation and 24 hours exposure without metabolic activation: all negative.

CYTOTOXICITY
In all in vitro genetic toxicity studies relevant cytotoxicity was not evident, except for a borderline result in preliminary toxicity testing for the mutation test with mouse lymphoma L5178 cells. In the latter pre-test, 24 h exposure to the precipitating concentrations of 1486 or 2971 µg/mL (without metabolic activation) produced relative suspension growth of 44 and 48%, respectively (relative to the suspension growth in concurrent vehicle controls).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The attained results do not necessitate any classification or labelling regarding mutagenicity according to REGULATION (EC) 1272/2008.