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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trifluoromethane
EC Number:
200-872-4
EC Name:
Trifluoromethane
Cas Number:
75-46-7
Molecular formula:
CHF3
IUPAC Name:
trifluoromethane
Details on test material:
- Purity: 99.9999%

Method

Target gene:
The objective of this in vitro assay was to examine the ability of H-30461 to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells in both the absence and the presence of a metabolic activation system (S9-mix). The assay with mouse lymphoma (L5178Y) cells detects forward mutations at the thymidine kinase (TK) locus on chromosome 11. The TK mutation test detects base pair mutations, frame shift mutations, small and larger deletions, and rearrangements of the relevant chromosome.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
TK-locus of cultured mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
0, 10, 20, 40, 60, 76 %
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: air
Controls
Untreated negative controls:
yes
Remarks:
76% N2, 19% O2, and 5% CO2
Negative solvent / vehicle controls:
yes
Remarks:
air
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the test duplicate cultures were exposed for 4 and 24 hours in the absence and 4 hours in the presence of S9-mix to 5 concentrations of the test substance ranging from 10 to 76%. All concentrations were evaluated for mutagenicity in the absence and presence of S9-mix.

DURATION
- Preincubation period: The L5178Y cells were grown in culture medium consisting of RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10 % v/v for growing in flasks, and 20 % for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin. The cells were cultured in a humidified incubator at ca. 37°C in air containing ca. 5 % CO2. Five to seven days prior to treatment, the cells were generated from a frozen stock culture from 6 November 2009 by seeding them in sterile, screw-capped tissue culture flasks (about 10000000 cells per flask: area ± 75 cm²) containing 50 mL culture medium (with 10 % horse serum). Fresh cultures of L5178Y cells were harvested from a number of culture flasks and suspended in culture medium (with 10 % horse serum), and the number of cells were counted. For the gene mutation test portions of ca. 3000000 and 5000000 L5178Y cells were used per culture in the absence and presence of S9-mix, respectively.

- Exposure duration: Since the test substance is a gas, cells in culture flasks were exposed in modular incubator chambers to various concentrations. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. The highest concentrations achievable was therefore 76%. Additional concentrations of 60%, 40%, 20% and 10% (all ±5%) were used in the chambers to expose the cells. Air without the test substance was be used as negative control. The test atmosphere was generated by mixing mass flow-controlled flows of oxygen, carbon dioxide, nitrogen and test substance. The mass flow controllers were calibrated by a volumetric flow meter. To ensure even distribution of the test atmosphere within the incubator chambers, these chambers were flushed with a volume of test atmosphere of at least five times the volume of the chamber. To prevent infection, a 0.45 μm filter (Millipore) was used. Thereafter, the supply and drain tubing of the chamber were blocked. The actual concentration of the test substance in the test atmospheres was measured twice in each chamber, once at the start of the experiment after flushing of the chamber and once at the end, just before opening the chamber.

- Cell treatment without metabolic activation: 5.0 mL culture medium without serum were added to ca. 3000000 L5178Y cells or 5000000 L5178Y cells (for 24 hours or 4 hours, respectively) in 5 mL culture medium (with 10 % horse serum) to a final volume of 10 ml. Two cultures treated with culture medium without serum were used as negative controls. One single culture treated with 100 μl MMS was used as positive control substance at a final concentration of 0.1 mmol/l. Duplicate cultures were used for each concentration of the test substance. The cells in culture flasks (area ± 25 cm2; without caps; ca. 0.4 cm layer culture medium covering cells) were exposed for 4h and 24 h at ca. 37°C and ca. 5 % CO2 in modular incubator chambers. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

- Cell treatment with metabolic activation: 4.0 mL culture medium without serum were added to 1 mL 20% (v/v) S9-mix (§7.3) and 5 mL culture medium (with 10% horse serum) containing ca. 5000000 L5178Y cells to a final volume of 10 ml. Two cultures treated with culture medium without serum were used as negative controls. One single culture treated with 100 μl MCA was used as positive control substance at a final concentration of 10 μg/ml. Duplicate cultures were used for each concentration of the test substance. The cells in culture flasks (area ± 25 cm2; without caps; ca. 0.4 cm layer culture medium covering cells) were exposed for 4 h at ca. 37°C and ca. 5 % CO2 in modular incubator chambers. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

DETERMINATION OF CYTOTOXICITY: The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG). The relative initial cell yield is the ratio of the number of cells after treatment to that of the vehicle control and is a measure for growth during treatment. The RSG is a measure for the cumulative growth rate of the cells 24 h and 48 h after treatment compared to untreated control cultures. The RTG is the product of the relative initial cell yield, the RSG and the relative colony-forming ability (‘cloning efficiency’) of the cells 48 h after treatment compared with negative control cultures, and is a measure for cytotoxicity that occurs in all phases of the assay.

After the treatment period, the cultures were checked for visible aberrant effects (eg. lysed cells), and the viability of the cells treated with the higher concentrations of test substance was checked. The medium containing the test substance, negative control or positive control was removed and the cells were washed twice with culture medium (with 10% horse serum). Finally, the cells were resuspended in culture medium (with 10% horse serum) and the number of cells was counted. The cell suspensions were diluted to 200000 cells per mL and the cultures were incubated for about 44-48 h at ca. 37°C and ca. 5% CO2 in a humidified incubator to allow nearoptimal phenotypic expression of induced mutations.

After 20-24 h and 44-48 h the number of cells of all remaining cultures was counted. After 20-24 h the cell suspensions were diluted, if required, to 200000 cells per mL and further incubated at ca. 37°C and ca. 5% CO2 as described above. After 44-48 h a portion of the cells was diluted in culture medium (with 20% horse serum) to 10 cells per mL for determining the cloning efficiency. The remaining cells were used for determining the frequency of TFT-resistant mutants. Portions (200 μl) of each dilution at 10 cells per mL were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5% CO2 in a humidified incubator. After this period the number of wells without growth of cells was counted and the cloning efficiency was determined using the zero term of the Poisson distribution. The ratio of the cloning efficiency of cells treated with the test substance or the positive control to that of the vehicle control yields the relative cloning efficiency (RCE). The ratio of the SG of treated cells to that of the vehicle control yields the relative suspension growth (RSG). The relative total growth (RTG) is adjusted for growth during treatment to obtain a measure for cytotoxicity that occurs in all phases of the assay. Reduction of the cell count after treatment, or of the RSG and of the RTG is a measure for the cytotoxicity of the test substance.

Gene mutation analysis: The frequency of TFT-resistant mutants and the cloning efficiency of the cells were determined 2 days after starting the test. The number of cells was counted and the cloning efficiency of the cells was determined. To determine the frequency of TFT-resistant mutants, the cell suspensions were diluted to a density of 10,000 cells per mL in culture medium (with 20 % horse serum) containing 4 μg TFT per ml. Portions (200 μl) of each dilution were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at ca. 37°C and ca. 5 % CO2 in a humidified incubator. After this period the number of wells without growth of cells was counted and the cloning efficiency in the TFT plates (Mutant cloning efficiency) was calculated. The mutant frequency (MF) per 1000000 clonable cells was calculated.

Evaluation criteria:
The following criteria were used to validate the data obtained in the gene mutation assay:
a) the average cloning efficiency of the negative controls should not be less than 60 % or more than 140 %.
b) the average suspension growth of the negative controls should be between 8 and 32.
c) the average mutant frequency of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1000000 clonable cells.
d) the mutant frequency of the positive controls should be higher than 400 TFT-resistant mutants per 1000000 clonable cells, and should be at least twice that of the corresponding negative control.
e) unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH and osmolar effects), the highest concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20%, or one data point between 1 and 10% and another between 20 and 30%.
Statistics:
The cloning efficiency of the cells was calculated from the total number of negative wells on the microtiter plates and the number of cells seeded per well. To assess the cytotoxic effects of the test substance or the positive controls on the cells, the initial cell yield after the treatment period, the relative suspension growth and the relative total growth to that of the vehicle negative controls were calculated. The cloning efficiency of the cells was used, together with the cloning efficiency on the TFT-containing plates, to calculate the mutant frequency. The mutant frequency was expressed as the number of TFT-resistant mutants per 1000000 clonable cells.

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:
other: slightly toxic at highest concentration
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Positive and negative controls
Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; an atmosphere of 76% N2, 19% O2, and 5% CO2 served as negative control. The negative controls were within the acceptance criteria and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls. Therefore, the test was considered valid.

Cytotoxicity
In the absence of S9-mix (24h treatment) the test substance was slightly toxic to the cells resulting in a reduction in the relative total growth (RTG). The mean RTG at the highest concentration evaluated in the absence of S9-mix (76%) was 71%. In the absence and presence of S9-mix after 4h treatment no toxicity was observed.

Mutagenicity
In both the absence and presence of S9-mix no increase in the mean mutant frequency (MF) by more than 88 or 126 mutants per 1000000 clonable cells, i. e. no equivocal or positive response, compared to the negative control was observed at any concentration level. See Tables 1 and 2.

Colony sizing
Since no increase of the mutant frequency was observed, colony sizing was not performed.

Any other information on results incl. tables

Table 1: Summary of the results in the absence of S9-mix

 

Conc (%)

Absence of S9-mix (24h)

Conc (%)

Absence of S9-mix (4h)

 

MF*

MF

RTG*

 

MF*

MF

RTG*

76

43

2

71

76

48

-20

90

60

49

8

84

60

69

0

91

40

53

13

74

40

70

1

101

20

43

2

92

20

64

-5

115

10

33

-8

100

10

60

-9

87

0

41

0

100

0

69

0

100

 

 

 

 

 

 

 

 

*Mean of duplicate cultures

MF = mutant frequency

MF = increase in mutant frequency

RTG = relative total growth

Table 2: Summary of the results in the presence of S9-mix

 

Conc (%)

Presenceof S9-mix (4h)

 

MF*

MF

RTG*

76

61

7

114

60

61

7

136

40

68

14

126

20

65

12

162

10

47

-7

142

0

54

0

100

 

 

 

 

*Mean of duplicate cultures

MF = mutant frequency

MF = increase in mutant frequency

RTG = relative total growth

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions used in this study, the test substance (Trifluoromethane) was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix).

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Executive summary:

The test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix) in a GLP, OECD guideline 476 study. One test was conducted. In the test, 5 duplicate cultures were treated for 4 hours in the presence and absence of S9-mix and 24 hours in the absence of S9-mix.

The highest nominal concentration of the test substance evaluated for mutagenicity was 76% both in the absense (4 and 24 hours) and presence (4 hours) of S9-mix. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2, the highest achievable concentration was 76%.

 

Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively; air served as negative control in both tests. The negative controls were within the acceptance criteria and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls. Therefore, the test was considered valid.

 

In the absence of S9-mix (24h treatment) the test substance was slightly toxic to the cells resulting in a reduction in the relative total growth (RTG). The mean RTG at the highest concentration evaluated in the absence of S9-mix (76%) was 71%. In the absence and presence of S9-mix after 4h treatment no toxicity was observed. 

 

In all tests evaluated for the mutant frequency in both the absence and presence of S9-mix no increase in mutant frequency (MF) by more than 88 or 126 mutants per 1000000 clonable cells, i. e. no equivocal or positive response, compared to the negative control was observed at any concentration level.

 

It was concluded that under the conditions used in this study, the test substance was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in both the absence and presence of metabolic activation (S9-mix).