Sulphur hexafluoride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The available studies on reproductive effects in rats by inhalation and intravenous exposure did not show any effect on reproductive performance and pre- and postnatal development and no maternal toxicity was seen at any exposure level tested.

Link to relevant study records

Reference

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other: GLP-compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

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

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

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

Dose / conc.:

50 000 ppm (nominal)

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.

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

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: 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.

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

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

Reproductive effects observed:

no

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.

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

50 000 ppm

Study duration:

subacute

Species:

rat

Additional information

In a reproduction screening study according to OECD 422, 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. Females were exposed for at least 2 weeks prior to mating, during mating and up to gestation day 19. No effect was observed on the investigated fertility and reproduction parameters. There was no effect on developmental parameters or on fetal development and no effects on the reproductive organs in males and females. No toxicologically relevant effects on early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. Moreover, no systemic effects were observed in the parents this study. 

The data from the inhalation study are complemented by the data from regulatory fertility and peri- and postnatal developmental studies in rats using the intravenous route (Willoughby, 1997 and Barrow, 1998) that have been conducted as part of the preclinical safety program of the pharmaceutical product SonoVue™ (BR1), an echo contrast agent based on stabilized sulfur hexafluoride (SF6) microbubbles. In these studies no effects on reproductive performance or developmental toxicity were observed at any exposure level. 

Effects on developmental toxicity

Description of key information

The available studies on developmental toxicity in rats and rabbits by inhalation and intravenous exposure did not show embryotoxic or teratogenic effects and no maternal toxicity was seen at any exposure level tested.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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

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.

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.

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

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

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

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).

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

Developmental effects observed:

no

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.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

20 000 ppm

Study duration:

subacute

Species:

rat

Additional information

In a recent OECD 414 study on Han Wistar rats, the effects of sulphur hexafluoride when administered during the organogenesis and fetal growth phases of pregnancy was examined. 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. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. The study revealed 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 also 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.

 

Furthermore, one study is available for inhalation exposure (Waalkens-Berendsen and H. Muijser, 2009). In this reproduction screening study according to OECD 422, 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. Females were exposed for at least 2 weeks prior to mating, during mating and up to gestation day 19. There was no effect on the developmental parameters or on fetal development and no effects on the reproductive organs in males and females. No toxicologically relevant effects on early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. Moreover, no systemic effects were observed in the parents in this study.

 

The data from the inhalation study are complemented by the data from regulatory developmental studies in rats and rabbits using the intravenous route (Willoughby, 1997) that have been conducted as part of the preclinical safety program of the pharmaceutical product SonoVue™ (BR1), an echo contrast agent based on stabilized sulfur hexafluoride (SF6) microbubbles. No maternal toxicity was observed in these studies and there was no evidence of embryotoxicity, teratogenicity or foetotoxicity at any exposure level. 

Justification for classification or non-classification

Based on the available data and in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for effects on fertility and developmental toxicity.