Registration Dossier
Registration Dossier
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EC number: 286-304-6 | CAS number: 85204-10-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- 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
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: chromosome aberration test in vitro
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix ( induced rat liver by Phenobarbital (PB) and β-naphthoflavone (BNF))
- Test concentrations with justification for top dose:
- Assay 1: (3 hours)
without S9-mix: 1500, 1000, 750, 500, 250, 120, 62.5 and 31.25 μg/mL
with S9-mix: 3000, 2500, 2000, 1000, 500, 250 and 125 μg/mL
Assay 2:
20 hours, without S9-mix: 3000, 2500, 2000, 1500, 1000, 500, 250 and 125 μg/mL
3 hours, with S9-mix: 1500, 1000, 750, 500, 250, 120, 62.5 and 31.25 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: appropriate for formulation and its dilution; compatible to the test system - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without S9-mix; dissolved in DMEM; 0.4 μL/mL (28-hour harvesting time) or 1.0 μL/mL (20-hour harvesting time) [
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with S9-mix; dissolved in 0,9% NaCl; 6.0 μg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:3 and 20 hours
- Fixation time (start of exposure up to harvest of cells): 20 and 28 hours
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.2 μg/mL)
STAIN (for cytogenetic assays): 5 % Giemsa solution
NUMBER OF CELLS EVALUATED: 200 cells (metaphases)/dose
DETERMINATION OF CYTOTOXICITY
- Method: determination of cell concentration by use of a haemocytometer
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- - Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures. - Statistics:
- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- Assay 1: without S9-mix: > 500 μg/mL; with S9-mix: > 250 μg/mL. Assay 2: No significant increase in the number of cells with structural chromosome aberrations with or without S9-mix.
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- without S9-mix: > 500 (250) μg/mL; with S9-mix: > 2000 (1500) μg/mL, in brackets values of Assay 2
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results (migrated information):
positive
In conclusion, WS400402 test item induced a significant level of chromosome aberrations in the performed experiments with and without metabolic activation. Therefore, WS400402 is considered clastogenic in this test system. - 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 internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.
- 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)
- 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 Wistar rats treated with phenobarbital and beta-naphthoflavone for enzyme induction.
- Test concentrations with justification for top dose:
- Range-finding test: 0; 10; 31.6; 100; 316; 1000; 2500 and 5000 μg/plate
Experiment 1: 0; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate
Experiment 2: 0; 5; 15.81; 50; 158.1; 500; 1581 and 5000 μg/plate - Vehicle / solvent:
- Distilled water
Justification for choice of solvent/vehicle:
Distilled water was a suitable vehicle for exposure to the test substance up to the maximum guideline recommended test substance concentration of 5000 μg/plate. - Untreated negative controls:
- yes
- Remarks:
- without and with S9 mix
- Negative solvent / vehicle controls:
- yes
- Remarks:
- without and with S9 mix
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene-diamine; sodium azide; 9-aminoacridine; methyl-methanesulfonate.
- Remarks:
- Positive control substances for tests without metabolic activation (S9 mix). All of them are well established reference mutagens.
- Untreated negative controls:
- yes
- Remarks:
- without and with S9 mix
- Negative solvent / vehicle controls:
- yes
- Remarks:
- without and with S9 mix
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene; Activity of S9 was additionally tested with 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:
- Range-finding test & Experiment 1: Standard Plate Incorporation Tests
Experiment 2: Pre-incubation Test
All tests were performed without and with metabolic activation (S9 mix)
Proportion of S9 fraction in the S9 mix was 10% v/v and of S9 fraction in the final medium ca. 2% v/v in all tests with metabolic activation.
The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:
Without metabolic activation (S9 mix):
4-nitro-1,2-phenylene-diamine: - 4 μg/plate, dissolved in DMSO: - strain: TA 98
Sodium azide: - 2 μg/plate, dissolved in distilled water: - strains: TA 100, TA 1535
9-Aminoacridine: - 50 μg/plate, dissolved in DMSO: - strain: TA 1537
Methyl-methanesulfonate: - 2 μL/plate, dissolved in distilled water: - strain: WP2 uvrA
With metabolic activation (S9 mix):
2-Aminoanthracene: - 2 μg/plate, dissolved in DMSO: - strains: TA 1535, TA 1537, TA 98, TA 100
2-Aminoanthracene: - 50 μg/plate, dissolved in DMSO: - strain: WP2 uvrA
In addition, adeqaute biological activity of the S9 batch used in the present study was confirmed by use of two mutagens, Aminoanthracene and Benzo(a)pyrene, that require metabolic activation by microsomal enzymes. - Evaluation criteria:
- The test substance is considered to exhibit mutagenic activity in this assay if the following criteria are met:
- a dose–related increase in the number of revertants and/or;
- a reproducible, biologically relevant positive response for at least one of the dose groups in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if in at least one of the five tested strains the number of reversion was more than twice higher than the reversion rate of the vehicle (solvent) control.
A test substance is considered non-mutagenic in this test if:
Neither a dose-related increase in the number of revertants nor a reproducible, biologically relevant positive response is evident in any of the dose groups, with or without metabolic activation.
The study was considered valid if:
- the number of revertant colonies of the vehicle (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests. - Statistics:
- The data were not statistically analysed. The study result was unequivocal.
- Species / strain:
- S. typhimurium, other: TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- in both experiments (Experiment 1 and 2)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- tested up to the recommended maximum concentration of 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- in both experiments (Experiment 1 and 2)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- confined to 1581 and 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Viability checked by a plating experiment in each test was satisfactory.
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'. Remarks: in the Range-finding test
- Conclusions:
- Interpretation of results (migrated information):
negative without and with metabolic activation (S9 mix) - 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
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine-kinase-locus (TK-locus)
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: 3 types of RPMI 1640 medium [Antibiotic-antimycotic solution 0,01 mL/mL, Pluronic-F68 0,5 mg/mL, Pyruvic acid 0,2 mg/mL, NaHCO3 2 mg/mL, L-glutamine 0,3 gm/mL] - with three conc. of horse serum (5, 10 and 20 % v/v, respective RPMI-5, -10, -20), medium with the highest conc. of horse serum RPMI-20 without Pluronic-F68.
Growth medium: RPMI-20
Expression-medium: RPMI-10
Selection-medium: RPMI-5
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- with S9 mix: rat liver, induced with Phenobarbital and β-naphthoflavone
- Test concentrations with justification for top dose:
- Based on the results of the preliminary toxicity experiment with doses up to 5000 μg/mL, the following test material concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation:
2500; 2187.5; 1875; 1562.5; 1250; 625; 312.5; 156.25; 78.125 and 39.06 μg/mL
Assay 1, 3-hour treatment without metabolic activation:
2500; 1250; 1093.75; 937.5; 781.25; 625; 468.75; 312.5; 156.25; 78.125 and 39.06 μg/mL
Assay 2, 3-hour treatment with metabolic activation:
2500; 2187.5; 1875; 1562.5; 1250; 625; 312.5; 156.25; 78.125 and 39.06 μg/mL
Assay 2, 24-hour treatment without metabolic activation:
1250; 1093.75; 937.5; 781.25; 625; 468.75; 312.5; 156.25; 78.125; 39.06 and 19.53 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: appropriate for formulation and its dilution; compatible to the test system.
A short solubility experiment showed, 250 mg/mL concentration was achievable using this vehicle (solvent). - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO / distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- assay without metabolic acitvation
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- dissolved in DMSO; final conc. in the experiments: 15 μg/mL for 3-hour treatment and 0.1 μg/mL for 24-hour treatment.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO / distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- assay with metabolic activation
- Positive control substance:
- cyclophosphamide
- Remarks:
- dissolved in DMSO, final conc. in the experiments: 4 μg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3h, 24h
- Expression time: 3 days
- Selection time: 2 weeks
SELECTION AGENT (mutation assays): Trifluorothymidine (TFT), final concentration of 3 μg/mL
REPLICATIONS: 2 cultures at each concentration
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, plating for viability
OTHER EXAMINATIONS:
- Scoring of large and small colonies was performed to obtain information on the mechanism of action of the test substance (gene or chromosome mutation). - Evaluation criteria:
- The test item is considered to be mutagenic in this assay if all the following criteria are met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle (solvent) control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding vehicle (solvent) control value. - Statistics:
- Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel 2000 software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- see Table 1: Summary of Results
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see Table 1: Summary of Results
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- PRELIMINARY EXPERIMENT
The highest concentration tested in the preliminary experiment was 5000 μg/mL. No insolubility of the test material was observed.
MAIN EXPERIMENT
In Assays 1 and 2, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment. - Conclusions:
- Interpretation of results (migrated information):
positive
A mutagenic effect of WS400402 was observed in the presence and absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Referenceopen allclose all
Table 1: Summarized results of the concentration selection cytotoxicity assays
|
Dose(μg/mL) |
Testgroup without S9-mix Relative survival (%) (Assay A/B) |
Testgroup with S9-mix (Assay A/B) |
Untreated control |
|
84/127 |
125/101 |
Vehicle control 1%(v/v) Distilled water |
|
100/100 |
100/100 |
Vehicle control 2% v/v Distilled water |
|
100/100 |
100/100 |
WS400402
|
5000 |
0/0 |
2/0 |
2500 |
0/0 |
41/18 |
|
1250 |
7/4 |
73/63 |
|
625 |
29/21 |
86/91 |
|
312,5 |
67/53 |
88/94 |
|
156,25 |
74/95 |
107/99 |
|
78,13 |
76/99 |
112/101 |
|
39,06 |
80/107 |
100/96 |
Time of Treatment/Sampling: 3h/20h
No insolubility of test material was detected at the end of the treatment period in the final treatment medium; there were no large changes in pH and osmolality either.
Table 2: Cytotoxicity results of the main experiments
|
Dose(μg/mL) |
Testgroup without S9-mix Rel. survival (%) Assay 1 3h/20h* |
Testgroup without S9-mix Rel. survival (%) Assay 2 20h/28h* |
Dose(μg/mL) |
Testgroup with S9-mix Rel. survival (%) Assay 1 3h/20h* |
Testgroup with S9-mix Assay 2 3h/28h* |
|
Vehicle control 2% v/v Distilled water |
|
100 |
100 |
|
100 |
100 |
|
WS400402
|
1500 |
0 |
0 |
3000 |
23 |
11 |
|
1000 |
2 |
1 |
2500 |
25 |
17 |
||
750 |
13 |
3 |
2000 |
40 |
48 |
||
500 |
37 |
12 |
1500 |
- |
36 |
||
250 |
73 |
41 |
1000 |
62 |
59 |
||
125 |
97 |
65 |
500 |
76 |
83 |
||
62,5 |
100 |
89 |
250 |
81 |
97 |
||
31,25 |
101 |
80 |
125 |
95 |
90 |
||
Positive control
|
- |
1μL/mL EMS 80 |
1μL/mL EMS 48 |
- |
51 |
6 μg/mL CP 52 |
*Treatment time/sampling
Table 3: Summary table of Chromosome Aberration Assay 1
Concentration (μg/mL) |
Time of Treatment |
Relative # Survival (%) |
Mean% aberrant cells### |
WS400402without metabolic activation (-S9) |
|||
Vehicle(solvent)control |
3h/20h |
100 |
1.0 |
1500 |
3h/20h |
0 |
NE |
1000 |
3h/20h |
2 |
NE |
750 |
3h/20h |
13 |
NE |
500 |
3h/20h |
37 |
18.1*** |
250 |
3h/20h |
73 |
2.0 |
125 |
3h/20h |
97 |
3.0 |
62.5 |
3h/20h |
100 |
NE |
31.25 |
3h/20h |
101 |
NE |
Positive control |
3h/20h |
80 |
12.4*** |
WS400402with metabolic activation (+S9) |
|||
Vehicle(solvent)control |
3h/20h |
100 |
2.0 |
3000 |
3h/20h |
23 |
NE |
2500 |
3h/20h |
25 |
NE |
2000 |
3h/20h |
40 |
60.0*** |
1000 |
3h/20h |
62 |
12.3*** |
500 |
3h/20h |
76 |
3.0 |
250 |
3h/20h |
81 |
11.0** |
125 |
3h/20h |
95 |
NE |
Positive control |
3h/20h |
51 |
96.8*** |
Vehicle (solvent) control: 2% (v/v) Distilled water Positive control (-S9):Ethyl methanesulfonate,1µL/mLPositive control (+S9): Cyclophosphamide, 6µg/mL
NE: notevaluated
#: compared to the vehicle (solvent) control
##:in the final treatment medium at the end of the treatment
###:excluding gaps
**: p<0.01comparing numbers of aberrant cells excluding gaps with corresponding vehicle (solvent)control
***:p<0.001comparing numbers of aberrant cells excluding gaps with corresponding vehicle (solvent)control
Table 4: Summary table of Chromosome Aberration Assay 2
Concentation (μg/mL) |
Time of Treatment/Sampling |
Relative # Survival (%) |
Mean% aberrant cells### |
WS400402without metabolic activation (-S9) |
|||
Vehicle (solvent) control |
20h/28h |
100 |
2.5 |
1500 |
20h/28h |
0 |
NE |
1000 |
20h/28h |
1 |
NE |
750 |
20h/28h |
3 |
NE |
500 |
20h/28h |
12 |
NE |
250 |
20h/28h |
41 |
1.5 |
125
|
20h/28h |
65 |
2.0 |
62.5 |
20h/28h |
89 |
1.0 |
31.25 |
20h/28h |
80 |
NE |
Positive control |
20h/28h |
48 |
50.8*** |
WS400402with metabolic activation (+S9) |
|||
Vehicle(solvent)control |
3h/28h |
100 |
3.0 |
3000 |
3h/28h |
11 |
NE |
2500 |
3h/28h |
17 |
NE |
2000 |
3h/28h |
28 |
NE |
1500 |
3h/28h |
36 |
7.0 |
1000 |
3h/28h |
59 |
4.5 |
500 |
3h/28h |
83 |
4.0 |
250 |
3h/28h |
97 |
NE |
125 |
3h/28h |
90 |
NE |
Positive control |
3h/28h |
52 |
68.2*** |
Vehicle (solvent) control: 2% (v/v) Distilled water
Positive control (-S9): Ethyl methanesulfonate,0.4µL/mLPositive control (+S9):Cyclophosphamide,6µg/mL
NE: not evaluated
#:compared to the vehicle (solvent)control
##:in the final treatment medium at the end of the treatmentment
###:excluding gaps
***:p<0.001comparing numbers of aberrant cells excluding gaps with corresponding vehicle (solvent) control.
None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in Assay 2 with or without metabolic activation although a dose related increase was observed across all the concentrations tested with metabolic activation.
Polyploid and endoreduplicated metaphases
Polyploid metaphases (1-4) were found in some cases in the vehicle (solvent) control, positive control or test material treated samples.No endoreduplicated metaphases were found in the two main tests except of one sample (in Assay 2 at 1500 μg/mL concentration with metabolic activation two endoreduplicated metaphases were found, all other values: 0).
Table 1: Summary of Results
| Assay | Highest concentration of WS400402 with evaluation possible / relative survival value | Statistically significant increase in the mutation frequency observed at concentration | |
Assay 1 (3h treatment with S9 mix) |
1875 μg/mL / 13% | >/= 1250 μg/mL |
|
Assay 1 (3h treatment without S9 mix) |
625 μg/mL / 15 % | 625 μg/mL* | |
Assay 2 (3h treatment with S9 mix) |
2187.5 μg/mL / 15% | >/= 1250 μg/mL |
|
Assay 2 (24h treatment without S9 mix) |
625 μg/mL / 9% | > 468.75 μg/mL** |
* Increased (but statistically or biologically not significant) mutation frequency was also detected at 468.75 μg/mL concentration.
** Increase at 468.75 μg/mL concentration did not exceed the global evaluation factor value
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Description of key information
In vivo genetic toxicity study (OECD Guideline 474 plus Comet Assay):
Three concentrations tested: 200, 400, 800 mg/kg b.w.; oral administration in Wistar rats (7 males/dose); three times administered - 48h, 24h and 4h prior preparation of the cell material. Micronucleus assay in bone marrow cells of tibia/femur; Comet assay in cells of the liver and of the small intestine.
Results: WS400402 exhibited no cytotoxicity in bone marrow and liver cells and did not induce micronuclei in bone marrow cells or DNA damage in liver cells. WS400402 was cytotoxic to cells of the small intestine, i.e. the first site of contact. The number of dead/apoptotic cells in this tissue was twice as high as the vehicle control value independent of the dose level. DNA damage of cells of the small intestine only was observed at the highest dose; this effect is considered as artefact caused by the very (too) high test substance concentration. It is concluded that WS400402 does not have genotoxic/mutagenic activity.
Bacterial Reverse Mutation Assay (OECD Guideline 471): negative
In vitro Mammalian Chromosome Aberration Test in Chinese Hamster V79 Cells (OECD Guideline 473): positive
In Vitro Mammalian Cell Gene Mutation Test (Mouse Lymphoma Assay) (OECD Guideline 476): positive
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- 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
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- For the Comet assay no internationally accepted guideline is available. This study was conducted according to the procedures indicated by the following recommendations: Tice, R.R. et al. (2000): Single Cell Gel/Comet Assay: Guidelines for ln Vitro and ln Vivo Genetic Toxicology Testing. Environmentaland Molecular Mutagenesis 35: 206-221 and Hartmann, A. et al. (2003): Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18 (1): 45-51
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Micronucleus Assay and Comet Assay
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS:
- Strain: rat (Wistar HsdCpb: WU)
- Source: Charles River Laboratories, Research Models and Services Germany GmbH,Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 271.8 g (SD +/- 6.7 g)
- Housing: in groups in cage type Makrolon Type IV, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: It shows relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w. - Details on exposure:
- oral administration
- Duration of treatment / exposure:
- 48h
- Frequency of treatment:
- All animals received three times (48h, 24h, 4h prior preparation) orally a single standard volume.
The animals of the positive control groups received the positive control substances once orally. - Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 400 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 800 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 7 male rats/dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- For the Micronucleus Assay: Cyclophosphamide (CPA)
- Administration: orally, once, 24h prior to preparation
- Doses / concentrations: CPA dissolved in sterile wate; 20 mg/kg b.w.
- Volume administered: 10 mL/kg b.w.
For the Comet Assay: Methylmethanesulphonate (MMS)
- Administration: orally, once, 4h prior to preparation
- Doses / concentrations: MMS dissolved in 0,9% NaCl solution; 25 mg/kg b.w.
- Volume administered: 10 mL/kg b.w. - Tissues and cell types examined:
- Micronucleus Assay: bone marrow cells of tibias/femur
Comet Assay: hepatocytes and cells from the small intestine - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity of WS400402 was performed with at least two animals per sex and test group under identical conditions as in the mutagenicity study concerning the following parameters: animal strain; vehicle; route, frequency, and volume of administration. Since no gender-specific differences in toxicity were observed, the main experiment was performed using male animals only treated with doses of 200, 400, and 800 mg/kg b.w. WS400402, respectively.
TREATMENT AND SAMPLING TIMES: Samples were taken at 24h and 48h - covering the interval in which maximum frequencies of micronuclei occur. This could be too late for the assessment of the DNA strand breaks, as these might have undergone repair. For this purpose a shorter additional treatment interval has to be considered in order detect damaged DNA in the correct time frame. For those reasons the animals were treated three times with three doses of the test item at intervals of 48 h, 24 h and 4 h prior to preparation.
DETAILS OF SLIDE PREPARATION: Three slides per organ of the animals were prepared with 10 % cell suspension and 90 % 0.7 % (w/v) agarose (low melting point agarose). 100 µL was applied per slide. The slides were cooled before being submerged in lysis buffer.
METHOD OF ANALYSIS:
Micronucleus Assay: At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei by manual inspection. The micronucleated PCE´s per 2000 PCE´s and the ratio of polychromatic erythrocytes to the total number of erythrocytes (NCE + PCE) are presented for each animal.
Comet Assay: DNA strand breaks were analysed using the alkaline single cell gel electrophoresis. One hundred cells per animal were evaluated on coded slides with a fluorescence microscope and the damage of each nucleus was measured and recorded by an image analysis programme (Comet Assay IV, Perceptive Instruments). - Evaluation criteria:
- Micronucleus Assay: A test item was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the tested dose levels.
Comet Assay: A test item was classified as mutagenic if it induced either a dose-related increase or a biologically relevant increase in the tail % intensity in a single dose group as compared to the historical control range. - Statistics:
- Micronucleus Assay: This was confirmed by means of the nonparametric Mann-Whitney test.
Comet Assay: Normally distributed data was analysed using a one-tailed student´s t-test. Not normal distributed data were analysed using a Mann-Whitney rank sum test. However, the primary point of consideration was the biological relevance of the results. - Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Micronucleus assay in bone marrow cells
- Toxicity:
- no effects
- Remarks:
- no cytotoxicity observed in bone marrow cells
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- Comet assay in liver cells
- Toxicity:
- no effects
- Remarks:
- no cytotoxicity observed in liver cells
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- the result of the Comet Assay in cells of the small intestine at the highest concentration (800 mg/kg b.w.) is disregarded (further discussion see in "applicant's conclusion")
- Toxicity:
- yes
- Remarks:
- cytotoxicity observed in cells of small intestine at each dose level without showing a dose-response effect
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 800 and 1250 mg/kg b.w.
- Clinical signs of toxicity in test animals: Severe toxic symptoms after the second treatment at the high concentration; the moribund animals were sacrificed. Animals treated three times with 800 mg/kg b.w. showed clear signs of toxicity, e.g. hunchback, ruffled fur. This concentration was taken as the highest one in the definitve study.
RESULTS OF DEFINITIVE STUDY
- Micronucleus assay: There was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei after administration of the test material and with any dose level used. The observed values in all test material treated groups are below the concurrent solvent controls; no micronuclei induced at none of the tested concentrations.
- Comet assay:
The comet assay on cells of the liver did not reveal a biologically relevant or statistically significant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Tail Intensity). All obtained test material group values were below or near to the values of the vehicle control group and additionally within the historical vehicle control data range.
The comet assay on cells of the small intestine, i.e. the first site of contact of cells with the substance, did not reveal an increase in DNA damage at 200 and 400 mg/kg b.w. of the tested item compared to the corresponding vehicle control on the evaluated parameter (% Tail Intensity). However, with the highest dose applied (800 mg/kg b.w.) an increased value of % Tail Intensity was observed as compared to the solvent control. This result is considered as artefact as discussed in "applicant's conclusion". The number of dead cells was twice as high as the vehicle control value in cells of the small intestine treated with test material - without showing a dose response effect.
Results for the comet assay in cells of the small intestine see Table 1 ("Any other information on results incl. tables"). - Conclusions:
- Interpretation of results (migrated information): negative
This in vivo genotoxicity test was performed because in two in vitro tests positive results were obtained for clastogenicity and for gene mutation.
In the micronucleus test-part of this study induction of micronuclei in bone marrow cells was not detected up to the highest dose. For evaluation of an in vivo potential for gene mutation in liver cells the Comet assay was performed instead of a test for unscheduled DNA synthesis (UDS). The positive control substance induced genotoxicity in liver cells whereas no effects were observed up to the highest dose of WS400402. From these results it can be stated that under the experimental conditions reported, WS400402, up to the maximum dose level tested, did neither induce micronuclei as determined by the micronucleus test with bone marrow cells nor primary DNA damage in the liver as determined by the Comet assay.
Next to these two endpoints in bone marrow cells and liver cells also cells of the small intestine, i.e. the first site of contact with the test substance, were investigated for DNA damage by the Comet assay. In these cells cytotoxicity was observed at each dose level whereas in bone marrow and liver cells no cytotoxicity was observed. The severity of cytotoxicity seen as apoptotic and dead cells was independent of the applied dose. At the highest dose, i.e. 800 mg/kg body weight applied by gavage, an increase of DNA damage was observed. At the two lower doses no effects were seen. However, this result of DNA damage is regarded as artefact for the following reason. The test substance WS400402 was applied by oral gavage at a concentration of 80 mg/ml with a dosing volume of 10 ml/kg body weight. This concentration is 40 times higher than the maximum recommended concentration of test substances in in vitro genotoxicity tests. A significant dilution of the test substance concentration in the stomach and small intestine does not occur. Thus, the cells were exposed to a very high concentration of WS400402 which already at a dose of 200 mg/kg b.w. (corresponding to 20 mg/ml) produced significant cytotoxicity to cells of the small intestine. Therefore, the observed increase of DNA damage at the highest dose of 800 mg/kg is considered an artefact due to too high test substance concentration and not as result of true genotoxic/mutagenic activity of WS400402. No effects in the cells of the small intestine were observed at the two lower dose concentrations. It is concluded that WS400402 does not have genotoxic/mutagenic activity.
Reference
Table 1a: Analysis of Dead Cell Index and % Tail Intensity in Cells of the Small Intestine
Test Group |
Dose (mg/kg b.w.) |
Dead Cell Index |
Comet Assyay Data |
|||
apototic/dead cells per 500 cells |
Standard Deviation |
Mean % Tail Intensity |
Standard Deviation |
Number of evaluable animals |
||
Negative Control |
0 |
15.3 |
11.1 |
4.55 |
1.7 |
7 |
WS400402 |
3x200 |
30.3 |
9.1 |
2.85 |
0.8 |
7 |
3x400 |
31.3 |
11.1 |
2.18 |
0.4 |
6* |
|
3x800 |
30.3 |
10.3 |
10.35 |
4.9 |
7 |
|
Methyl methane sulphonate |
1x25 |
32.0 |
12.5 |
21.98 |
6.2 |
7 |
* one animal scarified due to an application error
Table 1b: Biometry (Cells of the Small Intestine)
Negative controll versus test group |
Mean % Tail Intensity |
|
Dose of WS400402 |
Significance |
p |
3 x 200 mg/kg b.w. |
n.t. |
- |
3 x 400 mg/kg b.w. |
n.t. |
- |
3 x 800 mg/kg b.w. |
+ |
<0.011 |
1 x 25 mg/kg b.w. Methylmethanesulphonate |
+ |
<0.001 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vivo:
Justification for selection of genetic toxicity endpoint
The in vivo genetic toxicity study (OECD Guideline 474 plus Comet Assay with liver cells) is the most significant study, it is of high quality and reliability.
Justification for classification or non-classification
Based on the result of the in vivo genetic toxicity study it is concluded that WS400402 does not have genotoxic/mutagenic activity.
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