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EC number: 701-043-4 | CAS number: -
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Genetic toxicity: in vitro
Administrative data
- 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.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
Materials and methods
Test guidelineopen allclose all
- 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:
- in vitro mammalian chromosome aberration test
Test material
Constituent 1
Method
Species / strain
- 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
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix (induction of rat liver enzymes by Phenobarbital and β-naphthoflavone)
- Test concentrations with justification for top dose:
- Assay 1, 3h treatment with metabolic activation:
5000, 3750, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL
Assay 1, 3 h treatment without metabolic activation:
2000, 1000, 750, 500, 375, 250, 125, 62.5 and 31.25 μg/mL
Assay 2, 3h with treatment with metabolic activation:
2500, 2187.5, 1875, 1562.5, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/mL
Assay 2: 20h treatment without metabolic activation:
1000, 500, 375, 312.5, 250, 187.5, 125, 62.5 and 31.25 μg/mL
Assay 3, 3h with treatment with metabolic activation:
2000, 1500, 1000, 875, 750, 625, 500, 375, 250, 125, 62.5 and 31.25 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: appropriate for formulation and its dilution; compatible to the test system
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- without metabolic activation
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- Ethylmethanesulfonate (EMS) dissolved in DMEM, final concentration of 0.4 μL/mL (28-hour harvesting time) or 1.0 μL/mL (20-hour harvesting time)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- with metabolic activation
- Positive control substance:
- cyclophosphamide
- Remarks:
- Cyclophosphamide (CP) dissolved in sterile physiological saline solution (0.9% NaCl infusion), final concentration of 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.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see Table 1 and 2 ("Additional information on results")
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no changes
- Effects of osmolality: changes observed ≥ 3750 μg/mL in the experiment with metabolic activation (only in Assay 1).
- Insolubility: ≥ 312,5 and ≥ 375 μg/mL with and without metabolic acitvation, respectively. Insolubility was detected at the end of the treatment period in the final treatment medium.
Any other information on results incl. tables
Table 1: Summarized results of the concentration selection cytotoxicity assays A and B
|
Dose(μg/mL) |
Testgroup without S9-mix Relative survival (%) (Assay A*/B**) |
Testgroup with S9-mix (Assay A*/B**) |
Untreated control |
|
92/119 |
112/119 |
Vehicle control 1%(v/v) DMSO |
|
100/100 |
100/100 |
Vehicle control 2% v/v DMSO |
|
100/100 |
100/100 |
WS400104
|
5000 |
1/0 |
18/3 |
2500 |
23/0 |
62/2 |
|
1250 |
34/0 |
52/51 |
|
625 |
12/0 |
39/44 |
|
312,5 |
52/7 |
47/72 |
|
156,25 |
71/75 |
69/96 |
|
78,13 |
88/97 |
81/93 |
|
39,06 |
83/91 |
95/102 |
*Time of Treatment/Sampling: 3h/20h with and without activation
**Time of Treatment/Sampling: 20h/28h without activation, 3h/28h with activation
Table 2: Summary table of Chromosome Aberration Assay 1
Concentration (μg/mL) |
Time of Treatment / Sampling |
Relative Survival # (%) |
Mean aberrant cells### % |
WS400104 without metabolic activation (-S9) |
|||
Vehicle(solvent)control |
3h/20h |
100 |
3.0 |
2000 |
3h/20h |
19 |
NE |
1000 |
3h/20h |
25 |
NE |
750 |
3h/20h |
10 |
NE |
500 |
3h/20h |
32 |
5,0 |
375 |
3h/20h |
58 |
NE |
250 |
3h/20h |
64 |
4,5 |
125 |
3h /20h |
93 |
3,5 |
62.5 |
3h/20h |
92 |
NE |
31.25 |
3h/20h |
110 |
NE |
Positive control |
3h/20h |
84 |
11,0** |
WS400104 with metabolic activation (+S9) |
|||
Vehicle(solvent)control |
3h/20h |
100 |
3.0 |
5000 |
3h/20h |
0 |
NE |
3750 |
3h/20h |
27 |
2,7 |
2500 |
3h/20h |
0 |
NE |
1250 |
3h/20h |
0 |
NE |
625 |
3h/20h |
40 |
12,4** |
312,5 |
3h/20h |
87 |
3,5 |
156,25 |
3h/20h |
87 |
5,0 |
78,13 |
3h/20h |
94 |
|
39,06 |
3h/20h |
88 |
|
Positive control |
3h/20h |
66 |
96.8*** |
Vehicle (solvent) control: 1% (v/v) DMSO without (Assay –S9), 2% (v/v) DMSO (Assay +S9)
Positive control (-S9): Ethyl methanesulfonate, 1μL/mL; Positive control (+S9): Cyclophosphamide, 6μg/mL
NE: not evaluated
#: compared to the vehicle (solvent control)
##: in the final treatment medium at the end of the treatment
###: excluding gaps
**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
Table 3: Summary table of Chromosome Aberration Assay 2
Concentration(μg/mL) |
TimeofTreatment/Sampling |
Relative # Survival (%) |
Mean%aberrantcells### |
WS400104withoutmetabolicactivation(-S9) |
|||
Vehicle(solvent)control |
20h/28h |
100 |
3,0 |
1000 |
20h/28h |
0 |
NE |
500 |
20h/28h |
0 |
NE |
375 |
20h/28h |
0 |
NE |
312,5 |
20h/28h |
0 |
NE |
250 |
20h/28h |
15 |
NE |
187,5 |
20h/28h |
13 |
2.0 |
125 |
20h/28h |
57 |
1.0 |
62,5 |
20h / 28h |
91 |
2,0 |
31.25 |
20h/28h |
91 |
NE |
Positivecontrol |
20h/28h |
63 |
21,4*** |
WS400104withmetabolicactivation(+S9) |
|||
Vehicle(solvent)control |
3h/28h |
100 |
3.0 |
2500 |
3h/28h |
59 |
NE |
2187,5 |
3h/28h |
53 |
NE |
1875 |
3h/28h |
37 |
NE |
1562,5 |
3h/28h |
46 |
NE |
1250 |
3h/28h |
38 |
1,5 |
625 |
3h/28h |
47 |
6,0 |
312,5 |
3h/28h |
49 |
2,5 |
156,52 |
3h/28h |
87 |
2,0 |
78,13 |
3h/28h |
94 |
3,0 |
39,06 |
3h/28h |
75 |
NE |
Positivecontrol |
3h/28h |
58 |
12,6*** |
Vehicle (solvent) control: 1% (v/v) DMSO
Positive control (-S9): Ethyl methanesulfonate, 0.4μL/mL; positive control (+S9): Cyclophosphamide, 6 μg/mL
NE: not evaluated
#: compared to the negative (solvent control)
##: in the final treatment medium at the end of the treatment
###: excluding gaps
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
In Assays 1 and 2, none of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation except for 625 μg/mL concentration in Assay 1 with metabolic activation, in one replicate. This was not repeatable in Assay 2 (although elevated aberration frequency was observed at the same concentration), nor did the higher concentration assessed in Assay 1 with metabolic activation give a positive response. There was no evidence of any dose response in either assay.
Because of the equivocal results observed in Assay 1 with metabolic activation, an additional experiment with metabolic activation (Assay 3) was performed - with a more closely spaced concentration range, but otherwise using the same treatment and harvesting time as in Assay 1.
Table 4: Summary table of Chromosome Aberration Assay 3 with metabolic activation
|
Time of Treatment / Sampling |
Relative Survival#(%) |
Mean % aberrant cells### |
|
Vehicle(solvent)control |
3h/20h |
100 |
3.0 |
|
2000 |
3h/20h |
19 |
NE |
|
1500 |
3h/20h |
14 |
NE |
|
1000 |
3h/20h |
8 |
NE |
|
875
|
3h/20h |
4 |
NE |
|
750 |
3h/20h |
7 |
NE |
|
625 |
3h/20h |
34 |
2,5 |
|
500 |
3h/20h |
61 |
3,5 |
|
375 |
3h/20h |
81 |
2,0 |
|
250 |
3h/20h |
96 |
1,5 |
|
125 |
3h/20h |
89 |
NE |
|
62,5 |
3h/20h |
92 |
NE |
|
31,25 |
3h/20h |
88 |
NE |
|
Positive control |
3h/20h |
59 |
85,7*** |
Vehicle (solvent) control: 1% (v/v) DMSO
Positive control (+S9): Cyclophosphamide, 6μg/mL
NE: not evaluated
#: compared to the vehicle (solvent control)
##: in the final treatment medium at the end of the treatment
###: excluding gaps
***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control
It was concluded that the single result at 625 μg/mL with metabolic activation seen in a single replicate (not in the other replicate) was not reproducible in a repeat assay and did not show a dose response relationship. Hence this single result was not considered to represent an adverse effect of the test item.
The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid (1-11) and endoreduplicated (1-4) metaphases were found in some cases in the vehicle (solvent) control, positive control or test item treated samples in the performed experiments.
Applicant's summary and conclusion
- Conclusions:
- negative without and with metabolic activation (-/+ S9)
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