Sulphur hexafluoride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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

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:
- 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°C
- 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 (if applicable):

nose only

Vehicle:

other: air

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until mating occurred; all animals were mated within 5 days.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0
- After successful mating each pregnant female was caged individually for the birth and rearing of their pups. Dams were allowed to raise their litter until sacrifice on day 4 of lactation or shortly thereafter.
- Any other deviations from standard protocol: One sperm positive female of group 2 that turned out to be non-pregnant was killed 31 days after copulation.

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

Details on study schedule:

Age at mating of the mated animals in the study: ca. 12-13 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 000 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

50 215 ppm (analytical)

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, it was 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.

Examinations

Parental animals: Observations and examinations:

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: Yes
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.

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 AND COMPOUND INTAKE (if feeding study): yes
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.

FERTILITY AND REPRODUCTIVE PERFORMANCE
For each mating the following data were collected for each group:
- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sire
- number of pregnant females as demonstrated by the presence of implantation
sites observed at necropsy.
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live- and stillborn)
- number of live pups at day n
- number of pups lost
- number of litters lost entirely
- number of male pups at day n
- number of corpora lutea
- number of implantation sites
- number of lost implantations
- litter size

Sperm parameters (parental animals):

EPIDIDYMAL SPERM MOTILITY, COUNT AND MORPHOLOGY
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of 5 males/ group. For this purpose the left cauda was weighed and the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for these males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained. These smears were examined for morphology.

TESTICULAR SPERM COUNT
At necropsy, the left testis of 5 males/group was placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analyzed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated.

Litter observations:

PARTUTITION AND LITTER EVALUATION
At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

LITTER SIZE, SEXES AND WEIGHT
The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. The pups were individually weighed on days 1 and 4 of lactation. Mean pup weight was calculated per sex and for both sexes combined. The number of runts [defined as pup weight less than mean pup weight of the control group minus 2 standard deviations] was calculated.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Male animals were sacrificed after 29 days of exposure.
- Maternal animals: Females and their pups were sacrificed at or shortly after day 4 of lactation.

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
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
heart
intestines (small intestines, caecum, colon, rectum)
kidneys
larynx (3 levels, 1 level to include the base of the epiglottis)
liver
lung* (all lobes at one level, including main bronchi)
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
stomach
thymus
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 the treatment groups (group 2).

Postmortem examinations (offspring):

PATHOLOGY OF PUPS
No stillborn or pups that died during lactation were retrieved to perform a necropsy. At necropsy of the dams, at or shortly after day 4 of lactation, pups were examined externally for gross abnormalities and killed by decapitation and thereafter discarded.

Statistics:

- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live pups.
- Number of corpora lutea, implantation sites, live and dead fetuses or pups were evaluated by Kruskal-Wallis nonparametric analysis of variance
- Mortality data and data of the pathology of parent females were evaluated by the Fisher’s exact probability test.
- Sperm parameters were evaluated by ANOVA followed by Dunnett’s multiple comparison test (epididymal and testicular sperm count and numerical sperm motility parameters) or by Kruskal-Wallis non parametric ANOVA followed by Mann-Whitney U test (motility parameters expressed as a percentage and sperm morphology).

Reproductive indices:

The following reproductive indices were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
- mating index= (number of females mated/number of females placed with males) x 100
- male fertility index = (number of males that became sire/number of males placed with females) x 100
- female fertility index = (number of pregnant females/number of females placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated) x 100
- gestation index = (number of females with live pups or pups/number of females pregnant) x 100

Offspring viability indices:

The following parameters were calculated:
- live birth index = (number of pups born alive/number of pups born) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups on day n) x 100
- sex ratio day n = (number of live male fetuses or pups on day n/ number of live fetuses or pups on day n) x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/ number of corpora lutea] x 100
- number of lost implantations = number of implantations sites - number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born alive)/number of implantation sites] x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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:

effects observed, non-treatment-related

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:

effects observed, non-treatment-related

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

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Epididymal sperm motility, count and morphology: no differences were observed in the motility, count and morphology of the epididymal sperm at scheduled necropsy.
Testicular count: the testicular parachymal weight, the number of spermatozoa/g testicular parenchyma and the daily sperm production were statistically significantly decreased in the SF6-exposed group when compared to the control group. The results found in this study for the SF6-exposed group were well within the historical range and the decrease was therefore considered not to be related to exposure.

Reproductive performance:

no effects observed

Description (incidence and severity):

In each group 12 females were placed with males. No statistically significant difference in precoital time was observed; the mean pre-coital time for the control group was 2.8 and the SF6-exposed group 2.7 days.
The number of pregnant females and the number of males that became sires amounted to 12 and 11 for the control and the SF6-exposed group, respectively. All females delivered only liveborn pups.
The mating index, male fertility index, female fertility index, female fecundity index, and gestation index was 100% for the control group. The mating index, male fertility index, female fertility index, female fecundity index, and gestation index was 100, 92, 92, 92 and 100% for the SF6-exposed group, respectively.
The duration of gestation was 21.4 and 21.3 for the control and the SF6-exposed group, respectively.
No statistically significant difference was observed in the number of corpora lutea and implantation sites between the control and the SF6-exposed group. The number of corpora lutea was 11.4 and 11.9 for the control and the SF6-exposed group, respectively. The number of implantation sites was 10.6 and 10.0 for control and the SF6-exposed group, respectively.
Pre-implantation loss was 6.7 and 14.9 % for the control and the SF6-exposed group, respectively and was comparable between the groups.
Post-implantation loss was 5.5 and 4.5% for the control and the SF6-exposed group, respectively and was comparable between the groups.

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

302 687 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: no adverse effects at the highest tested concentration

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

On PN 1, the number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations) was statistically significantly increased in the SF6-exposed group when compared to the control group (10 runts, 3 litters of the SF6-exposed group and 1 pup of the control group). As no effect was observed on pup weight, the difference in number of runts was considered to be incidental. On PN 1, one pup of the control group showed a missing dead/tail tip. On PN 4, 2 runts (2 litters) were observed in the control group and 2 runts (1litter) were observed in the SF6-exposed group.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of litters with live born pups was 12 and 11 for the control and the SF6-exposed group, respectively. The number of pups delivered per litter in the control and the SF6-exposed group was 10.0 and 9.6, respectively. The number of live born pups amounted to 120 and 105 for the control and the SF6-exposed group, respectively and was comparable among the groups. No stillborn pups were detected. One pup of the SF6-exposed group died between postnatal day (PN) 1-4; pup mortality on PN 4 was 1% for the SF6-exposed group. No difference was observed in the sex ratio between the groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in pup weight and pup weight changes on PN 1 and 4 between the control and the SF6-exposed group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

One pup of the SF6-exposed group (pup 2 of dam 43) was missing on PN 4. No macroscopic observations were performed.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

302 687 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: No adverse effects on reproduction at the highest concentration tested.

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

For Sulphur hexafluoride, a No Observed Adverse Effect Level of 50,000 ppm for parental, reproductive and developmental toxicity was established in a combined inhalation repeated dose toxicity study and reproduction/developmental toxicity screening test (OECD 422, GLP).

Executive summary:

Sulphur hexafluoride has been studied in a GLP-compliant combined repeated dose inhalation toxicity study and 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 302687mg/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. Male and female animals were mated within the groups. Female animals were allowed to litter. The total litter size and numbers of each sex as well as the number of stillbirths, live and dead pups and grossly malformed pups were evaluated on days 1 and 4 of lactation. All pups were examined externally. The pups were individually weighed on days 1 and 4 of lactation. Females and their pups were sacrificed at or shortly after day 4 of lactation. Male animals were sacrificed after 29 days of exposure. Reproductive organs of 12 animals/sex/group were preserved and thereafter microscopically examined. The testes and epididymides were weighed of these animals were weighed. In addition for 5 animals/sex/group extra organs were weighed and preserved and thereafter microscopically examined. In these 5 male animals sperm analysis was also performed.

No mortalities or clinical signs were noted. Also no adverse effects were observed on body weight, food consumption,clinical chemistry and haematological parameters. The number of pregnant females and the number of males that became sires amounted to 12 and 11 for the control and the SF6-exposed group, respectively. All females delivered only liveborn pups. No effect was observed on the investigated fertility and reproduction parameters. No biological significant effects were observed with regard to the number of pups delivered per litter, the number of live born pupsand postnatal mortality. No stillborn pups were detected. No difference was observed in the sex ratio between the groups. In addition, SF6 did not induce a biological significant increase inthe number of runts (pup weight less than mean pup weight of the control group minus 2 standard deviations). No statistically significant differences were observed in pup weight and pup weight changes on PN 1 and 4 between the control and the SF6-exposed group. External examination did not reveal gross abnormalities. Based on these results, the NOAEC was set at the concentration of 50,000 ppm which corresponds to 302687 mg/m3.