Sulphur hexafluoride

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

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

Test animals

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: 322 g (males) and 194 g (females)
- 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

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

nose only

Vehicle:

other: air

Details on inhalation exposure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentrations of the test substance in the atmospheres were measured by photoacoustic 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 75 seconds) were transmitted and recorded on a PC. The daily mean response for each exposure unit was calculated by averaging all values collected during exposure.

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 minimum 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 000 ppm (nominal)

Remarks:

301631 mg/m3

Doses / concentrations 2

Dose / conc.:

50 215 ppm (analytical)

Remarks:

302687 mg/m3

No. of animals per sex per dose:

12/sex/concentration

Control animals:

yes

Details on study design:

- Dose selection rationale: because effects were not expected to occur, the Sponsor decided to perform a limit study at one high concentration (50,000 ppm)
- Rationale for animal assignment (if not random): computer randomization proportionately to body weight.
- Section schedule rationale (if not random): males after 29 days of exposure; females at day 4 of lactation or shortly after.
- Rationale for selecting satellite groups:
a) Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation.
b) Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken for haematology and clinical chemistry.
c) for 5 animals/sex/group extra organs were weighed and preserved and thereafter microscopically examined. In these 5 male animals sperm analysis and a micronucleus test were also performed.
d) On day 28 of the study, tail blood was collected of five SF6-exposed and five control male animals and the SF6 content in the blood was measured. In Section 7.1.1 further details are given about these measurements.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days and on all exposure days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. In the period before the start of exposure on Saturdays, Sundays, and public holidays, only one check per day was carried out. All abnormalities, signs of ill health, or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS AND NEUROBEHAVIOURAL OBSERVATIONS AND MOTOR ACTIVITY ASSESSMENT (ARENA TESTING, FOB AND MOTOR ACTIVITY)
Detailed clinical observations were conducted in all animals. Detailed clinical observations outside the home cage were performed prior to the first exposure and then once weekly.
Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation.

BODY WEIGHT: Yes
Body weights of male and female rats were recorded one day before the start of administration of the test substance at randomization, at the start of the study (day 0) and weekly thereafter during the premating period. Males were weighed once per week during the mating period until sacrifice. Females were weighed once per week during mating, and mated females were weighed on days 1, 7, 14 and 21 during presumed gestation and on day 1 and 4 of lactation.
In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
The food consumption was measured from day 0 onwards on the same days as the body weight was measured. The results were expressed in g per animal per day and g per kg body weight per day.

HAEMATOLOGY: Yes
On the day prior to the start of the mating period, 5 rats/sex/group were fasted overnight (water was freely available) and blood was taken, whilst under pentobarbital anaesthesia by orbital punction.
K3 EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated).

CLINICAL CHEMISTRY: Yes
On the day prior to the start of the mating period, 5 rats/sex/ group were fasted overnight (water was freely available) and blood was taken, whilst under pentobarbital anaesthesia by orbital punction. Blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation.

The following measurements were made in the plasma collected at necropsy:
alkaline phosphatase activity (ALP)
bilirubin (total)
aspartate aminotransferase activity (ASAT)
cholesterol (total)
alanine aminotransferase activity (ALAT)
triglycerides
gamma glutamyl transferase activity (GGT)
phospholipids
total protein calcium (Ca)
albumin sodium (Na)
ratio albumin to globulin (calculated)
potassium (K)
urea chloride (Cl)
creatinine inorganic phosphate (PO4).
glucose (fasting)

ANALYSIS OF SF6 in blood
On day 28 of the study, approximately 4 hours after the start of the exposure to SF6, tail blood was collected from 5 exposed and 5 control male animals. The concentration of SF6 in blood was determined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All male and female parent rats were sacrificed by exsanguination from the abdominal aorta whilst under pentobarbital anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after 29 days of exposure. Female animals were sacrificed at or shortly after day 4 of lactation.
Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde; except for the testes which was preserved in Bouin's fixative:
ovaries (after counting of the corpora lutea)
uterus (after counting of the implantation sites )
testes
epididymides,
seminal vesicles
prostate
all gross lesions

HISTOPATHOLOGY: Yes
For 5 animals/sex/ group, the following organs were preserved:
adrenals (also weighed)
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons) (also weighed)
heart (also weighed)
intestines (small intestines, caecum, colon, rectum)
kidneys (also weighed)
larynx (3 levels, 1 level to include the base of the epiglottis)
liver (also weighed)
lung* (all lobes at one level, including main bronchi) (also weighed)
lymphnodes from the hilar region
nasopharyngeal tissues (at least 4 levels; 1 level to include the nasopharyngeal duct and the Nasal Associated Lymphoid tissue (NALT)
oesophagus
spinal cord (cervical, mid-thoracic, and lumbar)
spleen (also weighed)
stomach (also weighed)
thymus (also weighed)
thyroid
trachea (at least 2 levels including 1 longitudinal section through the carina and 1 transverse section)

* The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation.

Microscopic examination was performed on the collected organs of all rats of the control (group 1) and treatment groups (group 2).

Other examinations:

See Section 7.6.2 for details on bone marrow micronucleus test and Sections 7.8.1 and 7.8.2 for details on the reproductive screening test.

Statistics:

The resulting data were analyzed using the methods mentioned below. P < 0.05 was considered as a level of significance.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one way analysis of variance (ANOVA)
- Functional observational battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data-total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 minutes each).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

One female of the control group was sparsely haired from GD 14 until sacrifice. One female of the SF6-exposed group showed encrustations of the eye from GD 2 until day 1 of lactation. The clinical observations observed in the parental animals are common findings in rats of this strain and age.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were observed in the mean body weight of the males during the premating and mating period until sacrifice. The mean body weight change of the SF6-exposed males was statistically significantly increased during the second week of the premating period. This effect was not considered to be treatment-related.
Mean body weight and body weight changes of the females during the premating, gestation, and lactation period was comparable among the control and the SF6-exposed group

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in the food consumption of the male and female animals during the entire study.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

In the males of the SF6-exposed group the percentage of neutrophils was statistically significantly increased and the percentage of lymphocytes statistically significantly decreased; no statistically significant difference was observed in the absolute numbers. These differences were small and were not considered as a toxicological effect. No other statistically significant differences were observed in haematology parameters among the control and the SF6-exposed group in the male and female animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The mean amount of total bilirubin was statistically significantly decreased in the males of the SF6-exposed group. This difference is likely to reflect normal biological variation and is well within the normal range. Furthermore, a decrease is considered of little importance.
In addition, a statistically significant increase was detected in the amount of PO4 of the female animals of the SF6-exposed group when compared to the control group; in the males a not significant increase was observed. Even substantial changes in level of PO4 in the extracellular fluid do not cause major effect on the body. Hence, the slight increment in PO4 was likely to be not relevant.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Weekly detailed clinical observations outside the home cage did not indicate an effect. A statistically significant decrease of the landing footsplay and a statistically significant increase of the motor activity were observed in male animals of the SF6-exposed group in comparison to controls. Those findings were considered incidental and are most probably due to the atypical control values and did not reveal an effect when compared to the historical range. Furthermore, no other differences in neurobehavioural testing were observed between the control and the SF6-exposed group.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and relative testes and epididymides weights of all male animals (12/group) were comparable among the control and the SF6-exposed group. The following organs weights were recorded in 5 animals/sex/group: adrenals, brain, heart, kidneys, liver, lung, spleen, stomach and thymus. The relative lung weight of the males of the SF6-exposed group was statistically significantly decreased. No effect was seen on the absolute and relative lung weight of the SF6-exposed females. In case of a treatment-related effect on the lungs, an increase on lung weight would be expected. In addition, no microscopic finding was observed on the lungs and the standard deviation was small compared to the control group. For these reasons this decrease in lung weight was considered as an incidental finding. All other absolute and relative organ weights were comparable between the control and the SF6-exposed group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination at necropsy revealed no exposure-related findings.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination of the testes, epidymides, seminal vesicle, prostate, uterus and ovaries of 12 animals/sex/group did not reveal any treatment-related effects. Microscopic examination of the adrenals, brain, caecum, colon, femur, heart, kidneys, larynx, liver, lung, nasal cavity, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, and tracheal/bronchial lymph nodes in 5 animals/sex/group did not reveal treatment-related histopathological changes in any of the sampled organs and tissues. The histopathological changes observed were considered unrelated to treatment because they were about equally distributed amongst the different groups or occurred in a single or few animals only.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Analysis of exposure conditions

Actual concentration:
The average (± standard deviation) of the daily actual mean concentrations as measured by photo-acoustic infrared analysis was 50,215 (± 142) ppm. This concentration was close to the target concentration of 50,000 ppm.

Nominal concentration:
The daily nominal concentration was calculated using the amount of test material used during the study (measured daily), the flows of humidified air and oxygen (measured weekly) and the time the test atmosphere was generated. The average (± standard deviation) of the daily nominal concentrations was calculated to be 48863 ppm. The average actual concentration in the same period was 50220 ppm indicating a generation efficiency of 103%.

Measurement of temperature, relative humidity carbon dioxide and oxygen content
Mean temperature (± standard deviation) during exposure was 22.1 (± 0.6) and 22.1 (± 0.5) °C for group 1 and 2, respectively. Temperature was below 20.0°C in group 1 once (during 2 minutes with a minimum of 19.9°C). Transgression of the upper limit of 24°C did not occur.

Mean relative humidity (± standard deviation) during exposure was 53% (±8) and 48% (±5) for group 1 and 2, respectively. Relative humidity exceeded the upper limit of 70% in group 1 on 17 days for a total period of 48 minutes (maximum: 74.5%). Relative humidity did not decrease below 30%.

Oxygen concentration was measured in the exposure chamber of group 2 on 2 June 2009 and was 20.7%. This did not differ from the ambient value in the laboratory.

Effect levels

Dose descriptor:

NOAEC

Effect level:

302 687 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose level tested.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of the current study, inhalation exposure to 50,000 ppm SF6 induced no adverse effects. Based on the results found in this study the No Observed Adverse Effect Level was 50,000 ppm SF6 for parental, reproductive and developmental toxicity.

Executive summary:

The sub-acute toxicity of sulphur hexafluoride was studied in GLP-compliant Combined Repeated Dose Inhalation Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed according to OECD Guideline 422 (TNO, 2009). Groups of 12 male and female Wistar rats were exposed daily to a limit (analytical) concentration of 302687 mg/m3 (target concentration of 50000 ppm) SF6 and air (control group) for 6 hours/day. 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. Prior to the end of the premating period, 5 rats/sex/group were fasted overnight and blood was taken for haematology and clinical chemistry. In addition, Functional Observational Battery (FOB) tests, including assessment of grip strength and sensory reactivity to stimuli of different types, and spontaneous motor activity measurements were performed in 5 males/group prior to the end of dosing and 5 females/group prior to day 4 of lactation. Males were sacrificed after 29 days of exposure; females at day 4 of lactation or shortly after.

The amount SF6 detected in the blood samples of the SF6-exposed animals ranged from 1.3-3.6 µg/ml blood (mean 2.3+0.7). No mortalities or exposure-related clinical observations were observed. Weekly detailed clinical observation and neurobehavioural testing did not indicate an exposure-related effect. No exposure-related differences were observed on body weight and food consumption. No relevant differences were observed on haematology and clinical chemistry between the control and the SF6-exposed group. The organ weights of the control and SF6-exposed group recorded in 5 animals/sex/group: adrenals, brain, heart, kidneys, liver, lung, spleen, stomach and thymus were comparable. Microscopic examination of the testes, epidymides, seminal vesicle, prostate, uterus and ovaries of 12 animals/sex/group did not reveal any treatment-related effects. Microscopic examination of the adrenals, brain, caecum, colon, femur, heart, kidneys, larynx, liver, lung, nasal cavity, rectum, small intestines, spinal cord, spleen, stomach, thymus, thyroid, trachea/bronchi, and trachea/bronchial lymph nodes in 5 animals/sex/group did not reveal treatment-related histopathological changes in any of the sampled organs and tissues. Based on these results, the NOAEC was set at the concentration of 50,000 ppm which corresponds to 302687 mg/m3.