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Toxicological information

Genetic toxicity: in vitro

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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:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. certificate)
Type of assay:
other: chromosome aberration test in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent

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
- 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
Controlsopen allclose all
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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
Relative survival (%)

(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
Rel. survival (%)

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

-

6 μg/mL CP

51

6 μg/mL CP

52

*Treatment time/sampling 

Table 3: Summary table of Chromosome Aberration Assay 1

 

Concentration

(μg/mL)

Time of Treatment
 /Sampling

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).

 

Applicant's summary and conclusion

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.