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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:
12 January 2016 to 03 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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan No. 287 - - Environment Protection Agency.
Eisei No. 127 - - Ministry of Health and Welfare.
Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Gene Mutation Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Apperance: Viscous gold coloured liquid
- Storage conditions: Room temperature in the dark

Method

Target gene:
Thymidine kinase (TK) locus.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were sub-cultured accordingly.
- Methods for maintenance in cell culture if applicable:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10 % donor horse serum (giving R10 media) at with 5 % CO2 in air. RPMI 1640 with 20 % donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Properly maintained: Yes. The stocks of cells are stored in liquid nitrogen at approximately -196 °C. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20 % donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically 'cleansed' against high spontaneous background: Yes. Periodically 'cleansed' against high spontaneous background: yes. The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Mutagenitcity Test: 3.91, 7.81, 15.63, 31.25, 62.5 and 125 (µg/mL) for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9. The maximum dose level used was limited by the presence of precipitate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: R0 media
- Justification for choice of solvent/vehicle: Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test material, the test material was accurately weighed and formulated in R0 medium prior to serial dilutions being prepared.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium
- Cell density at seeding:
4-hour exposure group with and without metabolic activation: 1 x 10^6 cells/mL in 10 mL aliquots
24-hour exposure group with and without metabolic activation: 0.3 x 10^6 cells/mL in 10 mL cultures

DURATION
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): Two days

SELECTION AGENT: Medium containing 4 µg/mL 5 trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: The exposures were performed in duplicate (A + B), both with and without metabolic activation.

MUTAGENICITY TEST
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm squared tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at 8 dose levels of the test material (0.98 to 125 µg/mL for all three exposure groups, vehicle and positive controls. 2 mL of S9-mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group) were added to each universal. The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test material. Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25 % of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates.
The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Evaluation criteria:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival (80 % toxicity), but no less than 10 % survival (90 % toxicity).
Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test material for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10 % are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
The experimental data was analysed using a dedicated computer program, Mutant 240C which follows the statistical guidelines recommended by the UKEMS. The statistical package used indicates the presence of statistically significant increases and linear-trend events.

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOTOXICITY TEST
The dose range of the test material used in the preliminary toxicity test was 1.95 to 500 µg/mL. These dose levels were selected based on precipitate observations form the solubility test. In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (% RSG) of cells treated with the test material when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was also evidence of marked reductions of %RSG values of cells treated with test material. A precipitate of the test material was observed at and above 62.5 µg/mL in all three exposure groups. In the subsequent mutagenicity experiments the maximum dose was limited by the incidence for precipitate.

MUTAGENICITY TEST
A summary of the results from the test is presented below. There was evidence of toxicity following exposure to the test material in all three of the exposure groups, as indicated by the % RSG and RTG values, which was more pronounced in the 24-hour exposure group. There was no evidence of any reduction in viability (%V), therefore indicating that no residual toxicity occurred in any of the three exposure groups. Optimum levels of toxicity were achieved in the 24- hour exposure group. In both 4-hour exposure groups the test material was exposed up to the maximum recommended dose based on precipitate observations. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

Any other information on results incl. tables

Table1. Summary of Results

Treatment

(µg/mL)

4-hours -S-9

4-hours +S-9

24 -S-9

%RSG

RTG

MF§

%RSG

RTG

MF§

%RSG

RTG

MF§

0

100

1.00

153.14

100

1.00

158.44

100

1.00

127.66

0.98 Ø

96

 

 

95

 

 

87

 

 

1.95 Ø

88

 

 

92

 

 

86

 

 

3.91

93

1.05

140.86

100

1.08

124.91

88

1.00

134.53

7.81

90

0.95

156.03

92

1.10

130.02

79

0.93

121.64

15.63

91

0.85

157.54

87

1.05

122.47

77

1.01

127.66

31.25

94

0.92

161.16

95

1.10

142.74

57

1.00

113.81

62.5

89

0.89

168.90

82

0.96

145.70

36

0.68

116.21

125

78

0.83

129.81

68

0.73

152.20

16

0.37

111.51

MF threshold for a positive response

279.14

284.44

253.66

Positive Control

EMS 400

64

0.44

1137.15

-

-

-

-

-

-

CP 1.5

-

-

-

72

0.61

724.32

-

-

-

EMS 150

-

-

-

-

-

-

38

0.38

937.71

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after treatment

CP = Cyclophosphamide

EMS = Ethylmethanesulphonate

Ø = Not plated for viability or 5-TFT resistance

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the test material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The GLP study was conducted in accordance with the standardised guidelines OECD 490 and EU Method B.17, US EPA OPPTS 870.5300 and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

One main Mutagenicity Test was performed and in this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at 8 dose levels in duplicate, together with vehicle (R0 media), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test material used in the main test was selected following the results of a preliminary toxicity test. The test material exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9. The maximum dose level used was limited by the presence of precipitate. A cloudy precipitate of test material was observed at and above 62.5 µg/mL at the end of the exposure period in all three exposure groups. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test material did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Under the conditions of the test, the test material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic in this assay