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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-02-17 to 2021-10-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
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
Purity: 99.9999%
Appearance: liquefied gas
Batch number: BWF200704
Supplier: Solvay Fluor GmbH
Shipping date: 10.12.2020
Packaging: 1 Cylinder
Expiry date: 01 December 2021
Storage conditions: at ambient temperature (15 to 25°C) during study use

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan®WIST rat
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 11 to 12 weeks old
- Weight at study initiation: 166 to 224 g
- Fasting period before study: No
- Housing: polycarbonate cages with a bedding of softwood bark-free fiber. A soft white untreated wood block and plastic tunnel, provided to each cage were used as environmental enrichment.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum except during the exposure
- Water (e.g. ad libitum):Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum except during the exposure
- Acclimation period: 5 days before commencement of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): monitored and maintained within the range of 20-24°C. On two occasions the temperature was out of the target range when it reached a maximum of 26°C, but each of these excursions lasted less than 1 hour.
- Humidity (%): monitored and maintained within the range of 40-70%
- Air changes (per hr): minimum 12
- Photoperiod (hrs dark / hrs light):12 hours light / 12 hours dark

IN-LIFE DATES:From: 06 April 2021 To:22 April 2021

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
snout only
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-through exposure chamber, modular construction in aluminum alloy comprising a base unit, a variable number of sections each having 20 exposure ports, and a top section incorporating a central aerosol inlet with a supplementary air inlet.
- Method of holding animals in test chamber: A separate chamber was used for each group. During exposure, the rats were held in restraining tubes with their snouts protruding from the ends of the tubes into the exposure chambers. Animal exposure ports not in use were closed with blanking plugs. The study animals were acclimated to the method of restraint, over a 3 day period preceding the first test item exposure. For each animal, training for dosing took place for 60, 180 and 360 minutes, with the restraint tubes being placed on an exposure chamber with exposure to air.
- Source and rate of air: Each exposure system was housed in a separate extract cabinet to avoid possible cross-contamination between groups.
- Air flow rate: 14 L/minute

TEST ATMOSPHERE
- Brief description of analytical method used: At least 3 samples for test groups (approximately 1, 3 and 5 hours into exposure) and one sample for the control group (approximately 1 hour into exposure) was withdrawn from each chamber, during each day of exposure.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples are collected in a gas bag and diluted with air to a nominal concentration within the range of 2500 to 15000 ppm, if required. 5 mL aliquots are injected onto GC.

GC conditions:
- Analytical column: ZB-5; 30 m x 0.32 mm ID, 0.25 µm film thickness
- Injection mode: split
- Injector temperature: 200°C
- Injection volume: 500 µL
- Detector type: Electron capture
- Detector temperature: 200°C
- Oven temperature: 100°C
- Approx. retention time: 0.8 to 1.2 minutes
- Carrier gas: Helium, 2 mL/min
- Split vent: Helium, 3 mL/min
- Make up: Nitrogen/air at 30 mL/min

Calibration:
The GC system was calibrated using external standards. Peak area data acquired by the data capture software using a 2nd order fit, with 1/x weighting, was subjected to least squares regression analysis.
Details on mating procedure:
Method: Time mated at the supplier to identified males of the same strain and source
Delivery to the test facility: Day 1 after mating
Day 0 of gestation: When positive evidence of mating was detected
Duration of treatment / exposure:
Females were exposed from GD6 to GD19 for 6 hours/day
Frequency of treatment:
Daily
Duration of test:
14 treatment days
Doses / concentrationsopen allclose all
Dose / conc.:
10 000 ppm (nominal)
Dose / conc.:
10 100 ppm (analytical)
Dose / conc.:
20 000 ppm (nominal)
Dose / conc.:
19 100 ppm (analytical)
No. of animals per sex per dose:
22 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target concentration levels for this study were selected on the basis of the results of a preceding combined repeated inhalation exposure toxicity study with the reproduction/developmental toxicity screening test in Wistar rats according to OECD 422 (TNO Report V8551) and a 90-day repeated dose inhalation exposure toxicity study in Wistar rats according to OECD 413 (TNO Report V20687) that were performed as limit tests at 50,000 and 20,000 ppm, respectively. The repeated dose toxicity studies did not result in any treatment-related changes in the parameters tested and the concentrations of 50,000 and 20,000 ppm were therefore considered as NOAEC for systemic and local toxicity. In addition, in the absence of data-based limits for concentrations in subchronic inhalation toxicity studies, the acute limit of the United Nations Globally Harmonized System of Classification and Labeling of Chemicals are used (i.e., up to a maximum concentration of 5 mg/L for aerosols, 20 mg/L for vapors, and 20,000 ppm for gases).
- Fasting period before blood sampling for (rat) dam thyroid hormones:
No overnight deprivation of food.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for all animals on Days 1, 3 and daily from Day 6 to 20 after mating

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 2-5, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals were killed on Day 20 after mating.
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded. Tissues were routinely preserved in 10% Neutral Buffered Formalin.
- Organs examined: Gravid uterine weight (including cervix and ovaries), thyroid.

THYROID HORMONE ANALYSIS:
Blood samples (1 mL) from all animals were collected at termination from the sublingual vein. No overnight deprivation of food was applied. Samples were kept at ambient temperature (15-25°C) for min. 30 minutes prior to centrifugation (2000g for 10 min. at 4°C). Two aliquots per animal were use: aliquot 1 for T3 and T4 determination, aliquot 2 for TSH determination.

T3 and T4 concentrations were measured by means of LC-MS/MS with use of a surrogate concentration range for quantification.
TSH levels were determined using the Luminex Hormone Magnetic Bead Panel with use of a calibration range for quantification.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Blood sampling:
- Plasma: No
- Serum: Yes, blood samples were collected at termination from all animals. To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons
- Blood sampling site: sublingual vein
- Volume collected : 1.0 mL
Fetal examinations:
Viable fetuses were dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.
Approximately 1/2 of the live fetuses in each litter were fixed in Industrial Methylated spirit and stained with Alizarin red for subsequent skeletal examination. The remaining fetuses were fixed in Bouin's fluid and examined for visceral abnormalities.
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights, ano-genital distance and thyroid weight:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel, 1959) was performed instead.
For the litter average ano-genital distance data, analysis of covariance was performed using the average pup body weight/fetal weight for each litter as the covariate (Angervall and Carlstrom, 1963), unless non parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in pup body weight/fetal weight which might influence the ano genital distance.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Indices:
- Pre-implantation loss were calculated as a percentage from the formula:
(No. of corpora lutea - No. of implantations) / No. of corpora lutea ×100 %
- Post-implantation loss were calculated as a percentage from the formula:
(No. of implantations - No. of live fetuses) / No. of implantations ×100 %
Sex ratios of the live fetuses were calculated as the percentage of males per litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs noted at the physical examination or associated with exposure.
Signs of wet fur and staining on head/eye were noted on return to home cage following exposure in animals of all groups, included controls. These signs were considered due to the method and duration of daily restraint and not test item related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test-item related effects on group mean body weight gain over Gestation Days 6 to 20.
The gravid uterine weight was unaffected by the test item, and there were no test item-related effects on body weight at termination, when adjusted for the gravid uterine weight. Detailed results are provided in Table 1-2 (see attached document).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 3 (see attached document).
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
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on circulating T4 or TSH levels. There were statistical significant differences from control in circulating T3 levels from animals exposed to 10100 and 19100 ppm but in the absence of a dose response these were considered not to be test item-related.
Detailed results are provided in Table 10-12 (see attached document).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related effects on the thyroid and parathyroid weights of females on Gestation Day 20.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on macropathology of females on Gestation Day 20.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related effects on the histopathology of the thyroids of females on Gestation Day 20.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4 (see attached document).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4 (see attached document).
Early or late resorptions:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4 (see attached document).
Dead fetuses:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4 (see attached document).
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
20 000 ppm
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed at the highest concentation tested

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 5 (see attached document).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4 (see attached document).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4 (see attached document).
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 4-5 (see attached document).
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 6 (see attached document).
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related major abnormalities.
At 19100 ppm, there was a slight increase in incidence of short supernumerary 14th ribs compared to the concurrent control, which was outside of the Historical Control Data (HCD) range and is thus considered test item-related. In addition, at 19100 ppm, there was a slight increase in incidence of medially thickened/kinked ribs, ossified cervical centra and brain haemorrhages compared to the concurrent control, but as these were within the Historical Control Data (HCD) range, were considered not test item-related.

At 10100 ppm there was a slight increase in incidence of medially thickened/kinked ribs and ossified cervical centra compared to the concurrent control, but as these were within the Historical Control Data (HCD) range, were considered not test item-related.

These findings are either transient in nature or a marker of maturity, and were considered not to be adverse.

Detailed results are provided in Table 7-9 (see attached document).

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest concentration tested

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The no observed adverse effect level (NOAEL) of Sulphur hexafluoride on systemic toxicity and embryofetal survival and development was considered to be 20,000 ppm.
Executive summary:



The study assessed the influence of Sulphur hexafluoride, an industrial chemical, on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar rat. The study was performed in accordance with OECD 414 and GLP.


Two groups of 22 females received sulphur hexafluoride at target exposure concentrations of 10000 or 20000 ppm by inhalation (gaseous) administration, from Day 6 to 19 after mating for 6 hours/day. A similarly constituted Control group received the air control. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination





The mean achieved atmosphere concentrations were 101 and 96% of target for Groups 2 and 3, respectively.


There were no test item-related effects on clinical signs, body weight, gravid uterine weight and adjusted body weight, food consumption or macroscopic pathology.


There were no test item-related effects on thyroid and parathyroid weights, or thyroid macropathology or micropathology or circulating T3, T4 or TSH levels.


There were no test item-related effects on embryofetal survival, as assessed by implantation rate, resorptions, sex ratio and litter size, or embryofetal development, as assessed by anogenital distance, placental, litter and fetal weight, and fetal abnormalities. A slight increase in minor fetal change of short supernumerary 14th ribs at 19100 ppm was considered transient in nature or a marker of maturity, and not adverse.


Conclusion


It was concluded that inhalation administration of Sulphur hexafluoride to female Han Wistar rats during the organogenesis and fetal growth phases of pregnancy at atmosphere concentrations of 10100 and 19100 ppm was without adverse signs of maternal toxicity or embryofetal survival and development.


A slight increase in the minor fetal change of short supernumerary 14th ribs at 19100 ppm was considered transient in nature or a marker of maturity, and not adverse.