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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.