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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 December 2015 to 16 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
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:
in vitro mammalian cell transformation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Stabilisation: in water undergoes hydrolysis
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch:13 October 2017
- Purity:92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability: instable after repeated contact to air

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dissolved in anhydrous DMSO and diluted prior to treatment. For dissolution of mixtures an ultrasonic treatment for 25 to 30 minutes at 37° C was necessary.
- Final dilution of a dissolved solid, stock liquid or gel: the maximum concentration was 2 mg/mL.

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: they are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus.
- Cell cycle length, doubling time or proliferation index: 10-12h doubling time.
- Cloning efficiency: more than 50%.
- Modal number of chromosomes: 40 ± 2 chromosomes

MEDIA USED: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank.
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction and NADP
Test concentrations with justification for top dose:
Pre-experiment for toxicity: 0.005, 0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0 and 2.0 mg/mL
Main experiment: 0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0 and 2.0 mg/mL
The selection of the concentrations used in the main experiment was based on data from the preexperiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Based on the results of the solubility test the test item was dissolved in DMSO.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 1% v/v
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):1 x 10^7 cells/11mL

DURATION
- Preincubation period: 1-3 days
- Exposure duration: 4h
- Expression time (cells in growth medium): 2 days
- Incubation time for cloning efficiency determination: 6 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 replications for negative and solvent controls. 2 plates per group in the cloning efficiency study, 4 plates per group in the mutagenicity study.

NUMBER OF CELLS EVALUATED: All cells were scored.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Rationale for test conditions:
In accordance with OECD 490 (17) "Media and culture conditions".
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of
126 mutants per 106 cells.
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
Non-parametric Mann Whitney test.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range.
- Precipitation: No precipitation of the test item was noted in the experiment.
- Other confounding effects: Growth inhibition was observed in the main experiment with metabolic activation at a single concentration of 0.5 mg/mL with a relative total growth (RTG) of 56.9. Considering that there was no evidence for a dose-response relationship this effect was considered as not biologically relevant.
In the main experiment without metabolic activation the relative total growth (RTG) was 88.0% for the highest concentration (2.0 mg/mL) evaluated. The highest concentration evaluated with metabolic activation was 2.0 mg/mL with a RTG of 88.3%.

RANGE-FINDING/SCREENING STUDIES: 9 concentrations [0.005, 0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0 and 2.0 mg/mL] were tested without and with metabolic activation. The experimental conditions in this pre-experiment were the same as described for the main experiment.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
EMS (Ethylmethanesulfonate): range 386.7- 2919.0, mean 719.5, SD 233.3.
MMS (Methylmethanesulfonate): range 378.2 - 2416.1, mean 837.3, SD 477.3.
B[a]P (Benzo[a]pyrene): range 303.6 - 1267.2, mean 650.3, SD 169.9.
- Negative control historical control data:
without S9: range 50.1 - 165.8, mean 87.9, SD 25.3
with S9: range 50.1 - 165.9, mean 84.7, SD 24.1
- Solvent control historical control data:
without S9: range 50.9 - 167.4, mean 91.6, SD 32.9
with S9: range 50.6 - 146.4, mean 84.0, SD 23.7
All mutant frequencies for negative, solvent and positive controls of the main experiment were found within the historical range of the test facility Eurofins Munich.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity is determined by measuring the colony forming ability and the growth rate of cultures.
- Other observations when applicable: clastogenicity: all dose groups were considered as not clastogenic in the main experiment with and without metabolic activation

Any other information on results incl. tables

Main Experiment - Mutagenicity Data,without metabolic activation

 

Cloning Efficiency (CE)

Mutagenicity Data

Test

Group

Concen­

tration

[mg/mL]

Plate

1e

Plate

2e

CEf[%]

Number of cultures / 96 wells

MFg

[mutants / 106cells]

IMFh

[mutants / 106cells]

Plate

1e

Plate

2e

Plate

3e

Plate

4e

Mean

C1

0

82

76

108.2

24

23

15

22

21.0

114.6

/

C2

83

73

104.6

19

18

28

24

22.3

126.7

/

S1

0

76

72

92.1

18

19

20

15

18.0

112.9

/

S2

81

80

114.0

22

14

16

15

16.8

84.5

/

3

0.010

74

79

99.6

19

25

22

18

21.0

124.2

25.6

4

0.025

78

69

90.7

13

12

13

19

14.3

88.9

-9.8

5

0.05

72

77

93.5

16

14

14

12

14.0

84.4

-14.3

6

0.10

83

81

120.3

19

19

22

11

17.8

85.5

-13.2

7

0.25

72

75

90.7

14

16

22

15

16.8

106.2

7.5

8

0.5

60

76

77.0

23

20

18

21

20.5

156.2

57.5

9

1.0

83

80

118.1

20

19

20

17

19.0

93.4

-5.3

10

2.0

78

77

102.9

17

20

22

21

20.0

113.6

15.0

EMS

300 pg/mL

63

72

75.9

68

61

70

73

68.0

819.5

720.8

MMS

10 pg/mL

57

56

55.5

43

54

45

38

45.0

575.9

477.2

Main Experiment - Mutagenicity Data,with metabolic activation

 

Cloning Efficiency (CE)

Mutagenicity Data

Test

Group

Concen­

tration

[mg/mL]

Plate

1e

Plate

2e

CEf[%]

Number of cultures / 96 wells

MFg

[mutants / 106cells]

IMFh

[mutants / 106cells]

Plate

1e

Plate

2e

Plate

3e

Plate

4e

Mean

C1

0

83

75

108.2

25

25

24

13

21.8

119.8

/

C2

78

77

102.9

17

14

26

20

19.3

109.6

/

S1

0

80

76

104.6

16

24

22

14

19.0

106.1

/

S2

81

77

108.2

16

20

16

20

18.0

96.1

/

3

0.010

76

71

90.7

18

15

22

26

20.3

131.5

30.4

4

0.025

78

70

92.1

18

20

14

16

17.0

106.0

5.0

5

0.05

72

78

95.0

21

16

18

23

19.5

119.8

18.7

6

0.10

84

78

116.0

16

18

14

16

16.0

78.6

-22.5

7

0.25

82

75

106.4

20

19

15

11

16.3

87.6

-13.5

8

0.5

67

72

80.5

11

17

29

21

19.5

143.4

42.3

9

1.0

77

78

102.9

25

14

21

20

20.0

114.1

13.1

10

2.0

74

80

101.2

17

17

12

20

16.5

93.5

-7.6

B[a]P

2.5 pg/mL

66

71

78.1

62

60

61

63

61.5

655.2

554.1

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The genetic toxicity of the test item was evaluated according to the OECD Guideline 490 with GLP. The potential to induce mutations at the mouse lymphoma thymidine kinase locus was studied using the cell line L5178Y. The selection of the test item concentrations used in the main experiment (0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0 and 2.0 mg/mL) was based on data from the pre-experiment. A negative control, a solvent control and three different positive controls were conducted. In the main experiment the relative total growth was 88.0% (without metabolic activation) and 88.3% (with metabolic activation) for the highest concentration (2.0 mg/mL) evaluated. In the main experiment no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). In the main experiment colony sizing showed no clastogenic effects induced by the test item. In conclusion, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.