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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:
July 28 - September 12, 2017
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)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay in mouse lymphoma L5178Y cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature protected from light
- Solubility and stability of the test substance in the solvent/vehicle: 5 mg/mL in 0.25% THF

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A stock solution of the test item was made with 0.25% THF. This stock solution was then diluted in RPMI and 5% HS.
- Final preparation of a solid: The test solution was then soniticated for 10 minutes at 37 degree C.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution

Method

Target gene:
TK 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 cell bank
- Cell cycle length, doubling time or proliferation index: 10-12 hr doubling time
- Whether whole blood or separated lymphocytes were used if applicable: lymphoma cells
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in RPMI 1640 complete medium.
- Modal number of chromosomes: 40

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
10, 20, 50, 100, 200, and 300 µg/mL
Top dose based on results of toxicity pre-experiment which found precipitation at concentrations of 400 µg/mL and above.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: THF was chosen due to the solubility of the test item in that solvent as compared to other solvents.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension of 11 mL RPMI medium and 5% horse serum
- Cell density at seeding (if applicable): 1 x 10E07

DURATION
- Preincubation period: 1-3 days
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days (8 days for cloning efficiency experiment)

SELECTION AGENT (mutation assays): RPMI with 20% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: all

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test substance is considered mutagenic if the following criteria were met:

induced mutant frequency of >= 126 mutants per 10E06 cells
dose-dependent increase in mutant frequency
increased occurrence of small colonies >= 40% of all colonies
Statistics:
Mean mutant frequency
Mean induced mutant frequency
p-value

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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at hightest dose level of 300 µg/mL

RANGE-FINDING/SCREENING STUDIES: A range-finding study was performed at concentrations of 10, 30, 100, 200, 400, and 1000 µg/mL with and without metabolic activation. Precipitation was seen at the 400 and 1000 µg/mL concentrations both with and without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Ethylmethanesulfonate (300 µg/mL): range - 318.7-2919.0, mean - 726.5, standard deviation - 203.5
Methylmethanesulfonate (10 µg/mL): range - 376.4-2416.1, mean - 763.4, standard deviation - 421.6
Benzo[a]pyrene (2.5 µg/mL): range - 303.6-1267.2, mean - 635.9, standard deviation - 167.8
- Negative (solvent/vehicle) historical control data:
Negative without S9: range - 50.1-170.3, mean - 87.9, standard deviation - 25.5
Negative with S9: range - 50.1-165.9, mean 85.1, standard deviation - 24.3

Any other information on results incl. tables

Mouse Lymphoma Assay Results – Without Metabolic Activation

Concentration

Relative Cloning Efficiency (%)

Relative Total Growth (%)

Mutant Frequency (mutants/10E06 cells)

Induced Mutant Frequency (mutants/10E06 cells)

Control 1

102.9

106.4

69.5

--

Control 2

98.0

100.3

69.5

--

Solvent Control

100.0

100.0

64.9

--

10 µg/mL

106.5

109.8

71.8

6.9

25 µg/mL

85.1

86.7

64.6

-0.3

50 µg/mL

102.9

97.3

86.5

21.6

100 µg/mL

87.7

81.2

73.9

9.0

200 µg/mL

104.7

86.6

69.0

4.2

300 µg/mL (Precipitate observed)

93.4

84.1

61.6

-3.3

Ethylmethanesulfonate*

91.9

73.0

649.0

584.2

Methylmethanesulfonate*

62.8

46.6

631.5

566.7

* Global Evaluation Factor exceeded and statistically significant increase in mutant frequency seen

Mouse Lymphoma Assay Results – With Metabolic Activation

Concentration

Relative Cloning Efficiency (%)

Relative Total Growth (%)

Mutant Frequency (mutants/10^6 cells)

Induced Mutant Frequency (mutants/10^6 cells)

Control 1

94.4

89.4

63.7

--

Control 2

107.0

105.6

63.7

--

Solvent Control

100.0

100.0

80.2

--

10 µg/mL

89.0

96.6

91.8

11.6

25 µg/mL

112.3

119.5

71.2

-8.9

50 µg/mL

91.6

84.8

68.4

-11.8

100 µg/mL

100.4

95.5

49.6

-30.6

200 µg/mL

98.9

96.2

72.7

-7.4

300 µg/mL (Precipitate observed)

114.2

102.7

60.4

-19.8

Benzo[a]pyrene*

90.3

64.4

702.5

622.4

* Global Evaluation Factor exceeded and statistically significant increase in mutant frequency seen

Applicant's summary and conclusion

Conclusions:
The test substance is non-mutagenic.
Executive summary:

The mutagenicity of the test substance was determined in an OECD Guideline 490 study. The mutagenicity of the test substance was determined at concentrations of 10, 20, 50, 100, 200, and 300 µg/mL both with and without metabolic activation. THF was used as a solvent. Negative, solvent, and positive controls were used. Precipitation was seen at the highest test concentration of 300 µg/mL. The test substance was not cytotoxic, nor was it mutagenic.