Sulphur hexafluoride

Genetic toxicity: in vitro

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:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sulphur hexafluoride

EC Number:

219-854-2

EC Name:

Sulphur hexafluoride

Cas Number:

2551-62-4

Molecular formula:

F6S

IUPAC Name:

sulphur hexafluoride

Test material form:

gas

Details on test material:

Name: SF6
Chemical name: Sulfur hexafluoride
Colour / appearance: colourless, odourless gas
CAS reg. number: 2551-62-4
Purity: 99.999 %
Batch number(s): BWF90327
Total quantity: 5 cylinders of each 40 kg
Molecular formula: SF6
Molecular weight: 146.07
Sublimation point: -63.9 °C
Melting point: -50.8 °C
Volatile: yes
Vapour pressure: 23700 hPa at 25 °C
Storage conditions: ambient temperature
Expiry date(s): 31 May 2019
Supplier: Solvay Fluor GmbH

Method

Target gene:

TK-locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

liver fraction of Aroclor 1254-induced rats for metabolic activation (S9-mix)

Test concentrations with justification for top dose:

76%, 60%, 40%, 20% and 10% (nominal concentrations)
The actual concentrations were measured at start and end of exposure and were all within 2% of the nominal concentrations. Analysis of SF6 in the medium after 24 hours exposure demonstrate maximum exposure of the cells.

Vehicle / solvent:

none

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: Methyl methanesulphonate (MMS), with S9: 3-methylcholanthrene (MCA)

Details on test system and experimental conditions:

In this study duplicate cultures were exposed for 24 hours and 4 hours in the absence and 4 hours in the presence of S9-mix to 5 concentrations of SF6 ranging from 10 to 76%. Finally, all 5 concentrations were evaluated for mutagenicity.

Generation and monitoring of the test atmosphere
The test atmosphere was generated by mixing mass flow-controlled flows of oxygen, carbon dioxide, nitrogen and SF6. The mass flow controllers (at the settings chosen) were calibrated by a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). 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. Samples of the test atmosphere were extracted at a known volume from the chamber using a calibrated gastight syringe (SGE 5 ml); the content of the syringe was injected into a gas sample bag filled with 9.3 nl clean, dry air (nl = litre under normal conditions, i.e. at 273.15K and 1013 hPa). The diluted samples (ranging in concentration from 50 to 120 ppm) were measured by photo acoustic infrared analysis (Bruel and Kjaer, Nearum Denmark) at a wavelength of 11.6 μm (filter UA0978). The responses of the analyzer (one response approximately every 60 seconds) were transmitted and recorded on a PC.
Before the start of exposure, the output of the infrared analyzer was calibrated. Test atmospheres carrying concentrations of 10, 20 and 60 vol % SF6 were generated by mixing mass flow controlled test atmospheres with oxygen, carbon dioxide, SF6 and nitrogen. The mass flow control units were calibrated as described above. Subsequently, respectively 4, 4.5 and 2.5 ml samples were extracted from these test atmospheres (in duplicate) and diluted in sample bags filled with 9.3 nl air. The diluted concentrations were calculated to be respectively 40, 90 and 150 ppm. The response Y (in ppm) of the infrared analyser was linearly related to the concentration C (in ppm) in the sample bags: R = 12.944 * C + 9.4448, with a coefficient of determination (R2) = 0.999. The dilution step was necessary because the sample volume (flow x time) necessary for the infrared analyser to obtain a stable output would be too large to extract from the relatively small incubator chamber. Moreover, it ensured linear operation of the infrared analyser.
The above mentioned relation was used to convert the reading of the infrared analyzer to the test atmosphere concentration of SF6 in the gas sample bag. The concentrations of SF6 inside the incubator chamber were calculated using the sample volume and the volume of dilution air in the gas sample bag.

Concentration analysis of test substance in medium
During 24 hours exposure, flasks filled with medium without cells were included in the exposure chambers at the 3 highest dose levels and the negative control.

Cell treatment without metabolic activation
In the assay without metabolic activation the cells were exposed to the test substance according to the following procedure; 5.0 ml culture medium without serum were added to ca. 3,000,000 L5178Y cells or 5,000,000 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 MMS was used as positive control substance at a final concentration of 0.1 mmol/l. Double cultures were used for each concentration of the test substance. The cells were exposed for 4 h and 24 h at ca. 37 °C and ca. 5 % CO2 in modular incubator chambers as described above.
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
In the assay with metabolic activation the cells were exposed to the test substance according to the following procedure; 4.0 ml culture medium without serum were added to 1 ml 20% (v/v) S9-mix and 5 ml culture medium (with 10% horse serum) containing ca. 5,000,000 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 MCA was used as positive control substance at a final concentration of 10 μg/ml. Double cultures were used for each concentration of the test substance. The cells were exposed for 4 h at ca. 37 °C and ca. 5 % CO2 in modular incubator chambers as described above.
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.

Assessment 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.

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 1,000,000 clonable cells was finally calculated as follows:
Mutant frequency (MF) = Mutant Cloning efficiency (MCE) / Cloning efficiency (CE) * 1,000,000

Analysis of results
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 1,000,000 clonable cells.

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells (Aaron et al, 1994; Clive et al., 1995). A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but smaller than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.
The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.
The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.
Both numerical significance and biological relevance were considered together in the evaluation.

Statistics:

No statistical analysis was performed.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Both in the absence and presence of S9-mix the test substance was not toxic to cells (no reduction in initial cell yield, suspension growth or relative total growth (RTG)). The relative total growth (RTG) in the absence of S9-mix at the highest concentration evaluated (76% SF6) was 108% and 92%, after 24 hours and 4 hours treatment, respectively (mean of duplicate cultures). In the presence of S9-mix the RTG at the highest concentration was 100% (mean of duplicate cultures).
In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. All data were within the range of the negative control and the historical background.
It is concluded that under the conditions used in this study, the test substance SF6 is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Applicant's summary and conclusion

Conclusions:

Sulphur hexafluoride did not induce gene mutations in the in vitro mouse lymphoma assay, at concentrations up to 76%, both with and without metabolic activation.

Executive summary:

In a GLP compliant study according to OECD guideline 476, SF6 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells (TNO, 2010). One assay was conducted in which 5 duplicate cultures were treated for 24 hours and 4 hours in the absence of S9-mix and 4 hours in the presence of S9-mix.

Since SF6 is a gas, cultures were exposed to the test substance in modular incubator chambers at ca. 37°C. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with nitrogen (N2). The nominal concentrations of the test substance were 76%, 60%, 40%, 20% and 10%. The actual concentrations were measured at start and end of exposure and were all within 2% of the nominal concentrations. Analysis of SF6 in the medium after 24 hours exposure demonstrate maximum exposure of the cells.

An atmosphere of 76% N2, 19% O2, and 5% CO2 served as negative control.

The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls. Both in the absence and presence of S9-mix the test substance was not toxic to cells (no reduction in initial cell yield, suspension growth or relative total growth (RTG)). The relative total growth (RTG) in the absence of S9-mix at the highest concentration evaluated (76% SF6) was 108% and 92%, after 24 hours and 4 hours treatment, respectively (mean of duplicate cultures). In the presence of S9-mix the RTG at the highest concentration was 100% (mean of duplicate cultures).In both the absence and presence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated.