Sulphur hexafluoride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sulphur hexafluoride did not induce gene mutations in the Ames test with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2 uvrA, and in the in vitro mouse lymphoma assay, at concentrations up to 76%, both with and without metabolic activation. Both studies were performed under GLP and according to OECD guidelines.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

TK-locus

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

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

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 all within 2% of the nominal concentrations.

Vehicle / solvent:

none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide, 9-aminoacridine, 2-nitrofluorene and N-ethyl-N-nitrosourea; with S9: 2-aminoanthracene and benzo(a)pyrene

Details on test system and experimental conditions:

Set-up
The plate-incorporation method with the histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98 and TA 100 and the tryptophan-requiring Escherichia coli mutant WP2 uvrA as indicator strains was applied. A preliminary test to assess the toxicity of the test substance was not performed. Therefore the toxicity test was incorporated in the mutagenicity assay.
Agar plates containing bacteria and S9-mix for the experiments with metabolic activation or sodium phosphate buffer for the experiments without metabolic activation were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations of the test substance. The chambers had a diameter of 27 cm and a height of 8.5 cm. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with N2. Five concentrations of SF6 were tested, namely 76%, 60%, 40%, 20% and 10% (all ±5%). Clean air without the test substance was used as negative control. Since only a limited number of plates could be exposed in each chamber, treatments with and without S9-mix were performed on two separate days.

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 Sm 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.

During the gene mutation test at the TK-locus of cultured mouse lymphoma (L5178Y) cells (study V8803/01) the concentration of the test substance was determined in the culture medium to demonstrate exposure of the test substance to the cells. The result of the analysis of the test substance is described in report V8803/01. Analysis of SF6 in the medium after 24 hours exposure demonstrate maximum exposure of the cells.

Mutation analysis
Fresh bacterial cultures were prepared by inoculation of nutrient broth with a thawed aliquot of the stock culture and subsequent incubation for approximately 10-16 h at 37°C while shaking. Briefly, the mutagenicity assay was carried out as follows. To 2 ml molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin for the S. typhimurium strains, and supplemented with 0.05 mM tryptophane for the E. coli WP2 uvrA strain), maintained at ca. 46°C, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate buffer 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The agar plates without lids were exposed to various concentrations of test substance or to the negative control (clean air) for 24 ± 0.25 hour at ca. 37 °C in the modular incubator chambers. After the exposure plates were removed from the chamber and further incubated for 24-48 hours at ca. 37 °C. For the positive controls also 0.1 ml of the positive control substance was added to the molten top agar. These plates were directly incubated for 48-72 hours at ca. 37 °C. Thereafter his+ and trp+ revertants were counted. Toxicity is defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control and/or the occurrence of pinpoint colonies.

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5% of the plates are lost through contamination or other unforeseen events.
A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates shows a concentration-related increase or if a reproducible two-fold or more increase is observed compared the negative controls.
A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.
Omission of a second test under these conditions is acceptable as a single test does not, or hardly ever results in false negative conclusions (TNO historical data and Kirkland and Dean, 1994).
Both numerical significance and biological relevance are considered together in the evaluation.

Statistics:

No statistical analysis was performed.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

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

Species / strain:

E. coli WP2 uvr A

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:

The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.
In both the absence and presence of S9-mix in all strains, SF6 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.
It is concluded that the results obtained with the test substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that SF6 is not mutagenic under the conditions employed in this study.

Conclusions:

Sulphur hexafluoride did not induce gene mutations in the Ames test with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia coli strain WP2 uvrA.

Executive summary:

SF6 was examined for mutagenic activity in the Ames test (according to OECD guideline 471 and GLP compliant) using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA (TNO, 2010). Since SF6 is a gas, agar plates containing bacteria were exposed to the test substance in modular incubator chambers for 24 ± 0.25 hours at ca. 37°C. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with nitrogen (N2). One bacterial reverse mutation test was performed with all strains in the absence and the presence of metabolic activation with five different concentrations of the test substance (76%, 60%, 40%, 20% and 10%). The actual concentrations were all within 2% of the nominal concentrations. Analysis of SF6 in the medium after 24 hours exposure demonstrate maximum exposure of the cells. Negative controls (clean air without the test substance) and positive controls were run simultaneously with the test substance.

The mean number of his⁺and trp⁺revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.

In both the absence and presence of S9-mix in all strains, SF6 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Sulphur hexafluoride did not induce any chromosomal damage in femoral bone marrow cells in a 28-days repeated dose inhalation toxicity study with a built-in micronucleus test (GLP compliant guideline study) with rats at a concentration of 302687 mg/m3.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 9-10 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) as environmental enrichment.
- Diet: cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum, except during the exposure
- Water: domestic mains tap-water suitable for human consumption, ad libitum, except during the exposure
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 40-70; reached 75.8 during one short period
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: gas

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Battelle tubes
Each exposure unit (Institute’s design) consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections of 20 ports each. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented. Animals were rotated each week with respect to the position in the column, viz. they were moved 5 places each time, and also weekly alternated between the upper, middle and lower sections.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The test atmosphere for group 2 was generated by mixing a mass flow controlled amount of gaseous test substance with a mass flow controlled stream of humidified compressed air. Because of the relatively high concentration of test substance, an additional mass flow controlled stream of oxygen was added to ensure a sufficiently high and, compared to the control group, equal oxygen concentration. The exposure unit for the control animals was supplied with a mass flow controlled stream of humidified compressed air only. The generated test atmospheres (total flow approximately 30 L/min for each exposure unit) were directed to the bottom inlets of the exposure units. At the top of the units the test atmospheres were exhausted. The animals were placed in the exposure units after stabilization of the test atmosphere.
The flows of humidified compressed air and oxygen at the settings chosen for the high dose group were used to calculate the flow of test substance necessary to reach the target concentrations. All flows were measured using volumetric flow meters (DryCal, Bios International Corporation, Butler, NJ, USA). Because the target concentration was given in ppm, the flows of test substance necessary to reach the respective target concentration follow directly from:
Test substance flow = total flow × concentration in ppm/1,000,000
The total flow consists of the flows of humidified air, oxygen and test substance vapour. The mass flow control unit for the test substance vapour was adjusted to the level computed using again the volumetric flow meters.
The settings (as initially chosen or computed) of the mass flow controllers (Bronkhorst, Hi Tec, Ruurlo, The Netherlands) were checked each morning at the start of generation, and subsequently at regular intervals during exposure (three times a day). The flows were 28 and 30 L/min for the control and exposed conditions, respectively.

TEST ATMOSPHERE
- Brief description of analytical method used: photoacoustic infrared analysis (Bruel and Kjaer, Nearum Denmark)
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hr/day
Males: for at least 2 weeks prior to mating, during mating and after the mating period at least until the minimm total exposure period of 28 days has been completed.
Females: for at least 2 weeks prior to mating, during mating and up to gestation day (GD) 19.

Frequency of treatment:

daily

Post exposure period:

None

Remarks:

Doses / Concentrations:
0 or 50000 ppm (0 and 301631 mg/m3)
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0 or 50215 (± 142) ppm (0 or 302687 (±857) mg/m3)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
0 or 48863 ppm (0 or 294772 mg/m3)
Basis:
nominal conc.

No. of animals per sex per dose:

5 males of the SF6-exposed, control and positive control groups were used for examination of micronuclei in the bone marrow

Control animals:

yes

Positive control(s):

mitomycin C (single exposure, approximately 24 hours before necropsy)
Route of administration: intraperitoneally

Tissues and cell types examined:

femoral bone marrow cells of the femur

Details of tissue and slide preparation:

BONE MARROW SAMPLING
At necropsy, femoral bone marrow cells of one of the femurs were collected from each rat assigned to the micronucleus test. The bone marrow cells were immediately collected into foetal calf serum and processed into glass drawn smears. Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald-Giemsa solution. The other unstained fixed smear was kept in reserve and discarded after completion of analysis.

MICROSCOPIC EXAMINATIONS OF BONE MARROW SMEARS
The bone marrow smears of the control and the SF6-exposed group and the positive control group were examined microscopically.

Evaluation criteria:

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.
The following criteria were used for the scoring of cells:
• A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
• A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
• A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.

The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Statistics:

Data on MPE and PE were subjected to a One Way Analysis of Variance (ANOVA) with factor treatment group (1, 2 and positive control). If the ANOVA yielded a significant effect (p<0.05), it was followed by non parametric tests (if residues were not normal distributed) or the Cochran approach (if spreading within the groups was different). These tests were applied to the negative control group (1) versus treatment SF6 -exposed (group 2) and the positive control group.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the micronucleus test in SF6-exposed male animals did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells or of cytotoxicity to the bone marrow.
Statistical analysis of the test results of the positive control indicated there were statistically significant (P< value 0.005) increases in the mean number of MPE in the positive control group compared with the control group. These increases were within the expected range. This indicates that the positive control substance Mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male and female rats. These results, together with the normal MPE/PE ratio in the control group, demonstrate the validity of the test system.

Conclusions:

Sulphur hexafluoride did not induce any chromosomal damage in femoral bone marrow cells in a 28-days repeated dose inhalation toxicity study with a built-in micronucleus test (GLP compliant guideline study) with rats at a concentration of 302687 mg/m3.

Executive summary:

The potency of SF6 to induce chromosomal damage was evaluated in a GLP-compliant 28-days inhalation study with reproductive and developmental toxicity screening, performed according to OECD guidelines 412, 422 and 474 (TNO, 2009). Groups of 12 male and female Wistar rats were exposed to a limit (analytical) concentration of 302687mg/m3 (target concentration of 50000 ppm) SF6 and air (control group) for 6 hours/day daily. Males were exposed for at least 2 weeks prior to mating, during mating and after the mating period at least until the minimum total exposure period of 28 days has been completed, while females were exposed for at least 2 weeks prior to mating, during mating and up to gestation day 19. Five males of the SF6-exposed, control and positive control (mitomycin C, single intraperitoneal administration, approximately 24 hours before necropsy) groups were used for examination of micronuclei in the bone marrow. The results of the micronucleus test in SF6-exposed male animals did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells or of cytotoxicity to the bone marrow. Statistical analysis of the test results of the positive control indicated there were statistically significant (P< value 0.005) increases in the mean number of MPE in the positive control group compared with the control group. These increases were within the expected range. This indicates that the positive control substance mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male and female rats. These results, together with the normal MPE/PE ratio in the control group, demonstrate the validity of the test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

SF6 was examined for mutagenic activity in the Ames test (according to OECD guideline 471 and GLP compliant) using the histidine-requiring Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the tryptophan-requiring Escherichia coli strain WP2 uvrA (TNO, 2010). Since SF6 is a gas, agar plates containing bacteria were exposed to the test substance in modular incubator chambers for 24 ± 0.25 hours at ca. 37°C. The atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test substance supplemented with nitrogen (N2). One bacterial reverse mutation test was performed with all strains in the absence and the presence of metabolic activation with five different concentrations of the test substance (76%, 60%, 40%, 20% and 10%). The actual concentrations were all within 2% of the nominal concentrations. Analysis of SF6 in the medium after 24 hours exposure demonstrate maximum exposure of the cells. Negative controls (clean air without the test substance) and positive controls were run simultaneously with the test substance.

The mean number of his⁺and trp⁺revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants nor a clearing of the background lawn of bacterial growth compared to the negative controls was observed.

In both the absence and presence of S9-mix in all strains, SF6 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.

 

In a study according to OECD guideline 476 and under GLP, 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.

 

The potency of SF6 to induce chromosomal damage was evaluated in a GLP-compliant 28-days inhalation study with reproductive and developmental toxicity screening, performed according to OECD guidelines 412, 422 and 474 (TNO, 2009). Groups of 12 male and female Wistar rats were exposed to a limit (analytical) concentration of 302687 mg/m3 (target concentration of 50000 ppm) SF6 and air (control group) for 6 hours/day daily. Males were exposed for at least 2 weeks prior to mating, during mating and after the mating period at least until the minimum total exposure period of 28 days has been completed, while females were exposed for at least 2 weeks prior to mating, during mating and up to gestation day 19. Five males of the SF6-exposed, control and positive control (mitomycin C, single intraperitoneal administration, approximately 24 hours before necropsy) groups were used for examination of micronuclei in the bone marrow. The results of the micronucleus test in SF6-exposed male animals did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells or of cytotoxicity to the bone marrow. Statistical analysis of the test results of the positive control indicated there were statistically significant (P< value 0.005) increases in the mean number of MPE in the positive control group compared with the control group. These increases were within the expected range. This indicates that the positive control substance mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male and female rats. These results, together with the normal MPE/PE ratio in the control group, demonstrate the validity of the test system.

In a study with insufficient documentation to make a reliable assessment (Sanotskii et al., 1981) the genotoxic potential of SF6 in the bone marrow of rats subjected to single or repeated exposure was

assessed. No positive responses were recorded after single exposure up to 250000 ppm or repeated exposure up to 12800 ppm SF6.

Justification for classification or non-classification

Based on the negative results of the available in vitro and in vivo studies and in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for genotoxicity.