Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Referenceopen allclose all

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
of 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 0; 5; 15; 50; 150; 500; 1500 and 5000 μg/plate
Experiment 2: 0; 50; 150; 500; 1500 and 5000 μg/plate
Vehicle / solvent:
Acetone
Justification for choice of solvent/vehicle:
A suspension in acetone was prepared suitable for exposure to the test substance up to the maximum guideline recommended test substance concentration 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:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
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:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
Positive control substances for tests with metabolic activation (S9 mix).
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% v/v in Experiment 1 whereas 20% v/v in Experiment 2.


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
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
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:
negative with and without metabolic activation (S9 mix)
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:
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
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.







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:
Preliminary toxicity tests were performed in the absence and presence of S9 mix to determine the concentrations at which the mitotic index would be reduced and at which cell toxicity was observed. Based on these results the concentrations for the main tests were determined. The following results were obtained:
3 hour treatment with test substance without S9 mix: at 540 µg/mL the mitotic index was reduced by 35% compared to control; at higher concentrations overt toxicity was observed.
3 hour treatment with test substance with S9 mix: at 1500 µg/mL the mitotic index was reduced by 51% compared to control; at higher concentrations overt toxicity was observed.
21 hour treatment with test substance without S9 mix: at 324 µg/mL the mitotic index was reduced by 30% compared to control; at higher concentrations overt toxicity was observed.

Concentrations prepared -S9, 3-hour treatment : 0*, 200, 250, 300*, 350, 400, 450*, 500, 550* μg/mL
Concentrations prepared +S9, 3-hour treatment: 0*, 50, 200, 400*, 500, 600, 700*, 800*, 900, 1000, 1200, 1300, 1400, 1500 μg/mL
Concentrations prepared -S9 , 21-hour treatment: 0*, 50, 100, 150*, 200, 250, 300*, 350, 400* µg/mL

* concentrations used for evaluation of metaphase analysis
Vehicle / solvent:
Acetone, the test substance formed a suspension in acetone at 250 mg/mL. At a concentration of 2500 µg/mL in tissue culture medium (1% of the acetone suspension) the test substance gave a visible precipitate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
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)
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: 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.

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.

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 recorded as a percentage of metaphases analysed.
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 concentration group and each positive control 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 the 1% 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
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both experiments, following 3 h or 21 h treatment
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance WS405777 did not cause statiscally significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration in the absence or presence of metabolic activation by S9 mix, when compared to the vehicle control.
Remarks on result:
other: not clastogenic
Conclusions:
negative with and without metabolic activation (-/+S9)
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:
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
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):
Cell culture media were obtained from a suitable supplier
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:
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):
4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL

MUTATION TESTS
Experiment 1, 3 h exposure (–S9):
Exposure concentrations: 0*, 50*, 150*, 200*, 250*, 300*, 325, 350, 400 μg/mL
Precipitation seen by eye at concentration of 250 µg/mL and above

Experiment 1, 3 h exposure (+S9):
Exposure concentrations: 0*, 50*, 150*, 300, 400*, 500*, 550*, 600, 625, 650 μg/mL
Precipitation seen by eye at concentration of 300 µg/mL and above

Experiment 2, 24 h exposure (–S9):
Exposure concentrations: 0*, 50*, 150, 300*, 400, 500*, 550*, 600*, 625, 650 μg/mL
Precipitation seen by eye at concentration of 150 µg/mL and above

* Mutant phenotype determination
Vehicle / solvent:
Acetone, the test substance formed a suspension in acetone at 250 mg/mL. At a concentration of 2500 µg/mL in tissue culture medium (1% of the acetone suspension) the test substance gave a visible precipitate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
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
True negative controls:
no
Positive controls:
yes
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
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The results obtained in response to the exposure of cell cultures to WS405777 in the absence or presence of metabolic activation by S9 mix did not demonstrate mutagenic potential. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
Remarks on result:
other: not mutagenic
Conclusions:
negative with and without 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 the three in vitro genotoxicity tests WS405777 is considered not to be genotoxic and does not warrant any classification regarding mutagenicity according to European classification rules [REGULATION (EC) 1272/2008].