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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:
in vitro mammalian chromosome aberration test

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

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
Relative survival (%)

(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

Concentration (μg/mL)

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)