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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria:

Key study: OECD 471. GLP study. The test item do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 with and without metabolic activation up to 5.0 µL/plate.

In vitro cytogenicity in mammalian cells:

Key study: OECD 473. GLP study. Read-across from the analogue ethyl lactate: The test item was determined to be not cytogenic it CHL/IU cells with and without metabolic activation up to 1200 µg/mL.

In vitro gene mutation study in mammalian cells:

Key study: OECD 490. GLP study. 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.

Link to relevant study records

Referenceopen allclose all

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
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
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.
Target gene:
Thymidine kinase locus
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.
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.
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

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

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 December 2015 to 29 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
According to OECD and EU method with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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
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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in DMSO and diluted prior to treatment.
Target gene:
Histidine-requiring gene in Salmonella thyphimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate. The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5 µL/plate was selected as the maximum concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, AppliChem Lot Nº4I013997
- Justification for choice of solvent/vehicle: The test item in water undergoes hydrolysis. The solvent was compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4- nitro-o-phenylene-diamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation test) and preincubation test

DURATION
- Preincubation period: Samples of each tester strain were grown by culturing for 12h at 37ºC in medium to achieve 10^9 cells/mL. For the pre-incubation method the tester strains were incubated during 60 min with the sterile buffer or the metabolic activation system and the test item preparation.
- Exposure duration:48h in the dark

SELECTION AGENT (mutation assays): L-histidine for S.typhimurium strains and tryptophan for E.coli strains

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED:all

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity could be detected by a clearing or diminution of the backgrown lawn or a reductionin the number of revertants down to a mutation factor of approximately <=0.5 in relation to the solvent control.
Rationale for test conditions:
According to the mentioned guidelines
Evaluation criteria:
A test was considered acceptable if for each strain:
-The bacteria demonstrate their typical responses to ampicillin
- The negative control plates with and without S9 mix are within the following ranges: TA98 (-S9: 13-48, +S9 13-61), TA100 (-S9:61-182, +S9:68-194), TA1535 (-S9: 4-35, +S9: 4-34), TA1537 (-S9: 2-27, +S9: 3-31) and Ecoli WP2 urvrA (-S9: 33-65, +S9:30-96).

A test item was considered as mutagenic if:
- A clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E.coli WP2 uvrA the number of reversions is at least twice as high
- if in the tester strain TA1535 an TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.
Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded necessary.
Key result
Species / strain:
S. typhimurium TA 98
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 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
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 1537
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:
E. coli WP2 uvr A
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
- Precipitation: No precipitation of the test item was observed in any tester strain used
- Other: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with the tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested (0.00316, 0.0100, 0.0316, 0.100, 1.0, 2.5 and 5.0 µL/plate. for toxicity and induction mutations with three plates each. The experimental conditions in this pre-experiment were the same as described for the main experiment.
Conclusions:
Under the experimental conditions reported, VL3 did not cause gene mutations in the genome of the tester bacteria strains used.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out usingSalmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). The test item was dissolved in DMSO, based on its solubility. Concentrations of 0.00316, 0.0100, 0.0316, 0.100, 1.0, 2.5 and 5.0 µL/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were as follows: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate. The exposition period was 48h in the dark. No positive effect was obtained in the examined bacterial strains with and without metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation in bacteria:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out usingSalmonella typhimuriumstrains TA98, TA100, TA1535 and TA1537 andEscherichia coliWP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). The test item was dissolved in DMSO, based on its solubility. Concentrations of 0.00316, 0.0100, 0.0316, 0.100, 1.0, 2.5 and 5.0 µL/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were as follows: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate. The exposition period was 48h in the dark. No positive effect was obtained in the examined bacterial strains with and without metabolic activation system.

In vitro cytogenicity study in mammalian cells:

Achromosome aberration test in Chinese hamster lung fibroblast cells (CHL/IU) was performed on the analogue ethyl lactate according to OECDE Guideline 473 (GLP study). Based on preliminary test, the study was performed up to 1200 µg/mL with and without metabolic activation. The test item was determined to be not cytogenic it CHL/IU cells with and without metabolic activation. Based on the read-across approach, VL3 is determined to be negative for cytogenicity.

In vitro gene mutation study in mammalian cells:

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.

Justification for classification or non-classification

Based on the available data (negative results in vitro), the test item is not classified for mutagenicity in accordance with CLP Regulation (EC) No. 1272/2008.