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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
TK locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: 10-12 h
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete
medium
- 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:
rat liver S9-mix
Test concentrations with justification for top dose:
Experiment I:
- 0.225, 0.250, 0.275, 0.300, 0.325, 0.350, 0.375, 0.400 μL/mL (without metabolic activation)
- 0.300, 0.325, 0.350, 0.375, 0.400, 0.425, 0.450, 0.475, 0.500 μL/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: THF was selected based on the solubility test of the test item; the solvent was compatible with the survival of the cells and S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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 RPMI medium with 5% horse serum
- Cell density at seeding: adjusted daily to 3 x 10E5

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: only negative and solvent controls were plated in duplicate; mutant frequency was counted in 4 plates for all treatment groups.

NUMBER OF CELLS EVALUATED: four 96-well plates at a density of approximately 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth (SG; the number of times the cell number increased from the starting cell density); relative total growth (RTG; the product of the relative suspension growth [RTG] and the relative cloning efficiency [RCE])
Rationale for test conditions:
The assay was considered acceptable if the following criteria were met:

1. At least 3 out of 4 of the 96-well plates were scorable;
2. The cloning efficiency of the negative and/or solvent controls was between 65-120%;
3. The spontaneous mutant frequency in the negative and/or solvent controls was between 50 to 170 mutant per 10E6 cells;
4. The cell number of negative and/or solvent controls increased between 8 to 32 fold during the 2-day growth period; and
5. Positive controls responded appropriately and produced either at least 300 mutants per 10E6 cells with at least 40% of the colonies being small OR indcued a small colony mutant frequency of at least 150 mutants per 10E6 cells. The RTG for positive controls also must be greater than 10%.
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 10E6 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.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.

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:
Statistical significance at the 5% level (p < 0.05) was evaluated for mutant frequency by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG was 10.1% for 0.400 mg/mL, -S9, and 9.6% at 0.500 mg/mL, +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutant frequencies without metabolic activation were not statistically significantly increased over the solvent controls. With metabolic activation, there was a statically significant increase in mutant frequencies over the solvent controls at 0.350, 0.375, and 0.475 mg/mL; however, these increases were not considered treatment related because there was no evidence of a dose-relationship.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH dectected was within the normal physiological range.
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: test item is water-reactive
- Precipitation: No precipitation was observed in the main experiment; precipitation was observed in the pre-experiment at a concentration of 2 mg/mL.

RANGE-FINDING/SCREENING STUDIES: A pre-experiment was conducted at concentrations up to 2 mg/mL.

HISTORICAL CONTROL DATA
- Positive historical control data: EMS (300 μL/mL): 726.5±203.5; MMS (10 μL/mL): 763.4±421.6; B[a]P (1.5 μL/mL): 535.5±152.5
- Negative (solvent/vehicle) historical control data: Negative control, -S9: 87.9±25.5; Negative control, +S9: 85.1±24.3; THF, -S9: 105.9±30.0; THG, +S: 10.6±23.5

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative total growth (RTG)
- Other observations when applicable: Growth inhibition was observed both with and without metabolic activation. Without metabolic activation, the relative total growth (RTG) was 10.1% for the highest concentration (0.400 mg/mL); with metabolic activation, the RTG was 9.6% at 0.500 mg/L.

Any other information on results incl. tables

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration
[mM]

Relative cloning efficiency [%]

Relative Total Growth [%]

Mutants per 10E+06 surviving cells

Mutation factor

Negative 1

96.5

87.7

90.4

/

Negative 2

77.0

71.7

113.2

/

Solvent (THF) 1

100.0

100.0

70.1

/

Solvent (THF) 2

65.6

/

0.300

101.3

95.9

82.9

15.1

0.325

103.0

98.5

96.6

28.8

0.350

93.6

83.9

114.8*

47.0

0.375

90.7

82.2

115.7*

47.9

0.400

101.3

72.2

60.4

-7.4

0.425

103.0

18.9

65.0

-2.9

0.450

104.7

33.3

66.8

-1.0

0.475

88.0

25.1

102.0*

34.2

0.500

96.5

9.6

54.1

-13.7

B[a]P, 3.5 µg/mL

90.7

61.3

537.1*

469.2

B[a]P  Benzo[a]pyrene

*         Statistically significant

 

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration
[mM]

Relative cloning efficiency [%]

Relative Total Growth [%]

Mutants per 10E+06 surviving cells

Mutation factor

Negative 1

93.0

91.2

91.8

/

Negative 2

99.2

106.1

72.6

/

Solvent (THF) 1

100.0

100.0

114.7

/

Solvent (THF) 2

82.8

/

0.225

93.0

78.0

86.5

-12.2

0.250

104.3

92.4

52.6*a

-46.2

0.275

87.4

68.1

90.5

-8.2

0.300

113.9

93.6

85.9

-12.9

0.325

94.5

67.4

88.9

-9.9

0.350

81.1

40.9

84.8

-14.0

0.375

84.8

34.6

109.4

10.7

0.400

73.2

10.1

99.0

0.3

EMS, 300 µg/mL

90.2

71.3

595.3*

496.5

MMS, 10 µg/mL

70.1

55.5

690.4*

591.7

EMS    Ethylmethanesulfonate

MMS   Methylmethanesulfonate

*         Statistically significant

a         Significantly decreased compared to the solvent control, therefore not relevant for interpretationof results

Applicant's summary and conclusion

Conclusions:
In an OECD 490 study, the test material is not considered to be mutagenic in an in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells both with and without metabolic activation.