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Genetic toxicity in vitro

Link to relevant study records

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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:
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
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

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).

 

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:
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
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 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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.


Summary tables of survival, viability, and mutagenicity results are attached as background material.

Additionally, upon request of ECHA the full study report is attached.

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

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.
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. However, no DNA damage of cells of the small intestine was observed up to the highest dose

Link to relevant study records
Reference
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
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. certificate)
Type of assay:
other: Micronucleus Assay and Comet Assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals 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:
Comet assay in cells of small intestine
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. The results table is attached as background material.
- 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 up to the highest dose level although at all dose levels cytotoxicity was observed. The results tables are attached as background material. PLEASE NOTE: in the results tables there is a hand written correction of the claculated group mean value of high dose animals. This wrong value was reported in the final report and interpreted as DNA damage in intestinal cells. However, the correct group mean value shows that there was no DNA damage. Discussion with the laboratory to revise the report were started on 26 June 2019.
Conclusions:
Interpretation of results: 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 with the test substance WS400402 no effects were observed up to the highest dose. 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 in 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. Whereas in bone marrow and liver cells no cytotoxicity was observed, in cells of the small intestine cytotoxicity was observed at each dose level. However, no DNA damage was observed up to the highest dose level.
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.