Sulphur hexafluoride

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

GLP compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Objective of study:

absorption

excretion

GLP compliance:

yes (incl. QA statement)

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:

solid - liquid: suspension

Radiolabelling:

no

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on species / strain selection:

The rabbit was chosen as a model because it best fulfils both analytical and pharmacokinetic requirements of this study. Strictly controlled analytical conditions are necessary to recover the gas, SF6, at trace levels (ng level) in blood, in exhaled air, and in urine. SF6 concentrations can only be quantified if blood samples are sufficiently large (1-3 ml/sampling). Moreover, serial sampling of blood is necessary to establish blood kinetics.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS - New Zealand White rabbits
- Source: Geneva University Medical Centre (CMU) breeding colony, Switzerland
- Weight at study initiation: 3.2 ± 0.3 kg
- Housing: Stainless steel cages
- Diet: Rabbit food (UAR NQ 112 C, Indulab Buchs, Switzerland)
- Water: tap water "ad libitum"

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2°C
- Humidity (%): 50 ± 10% rH
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 May 1993 To: 16 July 1993

Administration / exposure

Route of administration:

intravenous

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
SonoVue™ (BR1) is a suspension of stabilized SF6 microbubbles in saline (0.9% sodium chloride). BR1 was supplied as a sterile lyophilized powder (50 mg) in a gaseous atmosphere (SF6) in 20-mL vials. For the preparation of SonoVue™, air was first removed from septum-sealed vials using a water pump vacuum and replaced with SF6 from a gas cylinder at a pressure of 1 bar. Just prior to administration, the test article was reconstituted directly in septum sealed vials by adding 10 ml saline (NaCl 0.9%, w/v) to the lyophilized preparation using standard clinical aseptic techniques. The concentration SF6 is 7.0 μL/ml (approx. 40 µg/ml) after reconstitution.

Duration and frequency of treatment / exposure:

Single intravenous administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.3 other: ml/kg

Remarks:

(12 µg SF6/kg)

Doses / concentrations 2

Dose / conc.:

1 other: ml/kg

Remarks:

(40 µg SF6/kg)

No. of animals per sex per dose / concentration:

6 males/dose group;
5 males/control group

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels of 0.3 ml/kg (12 µg SF6/kg) and 1.0 ml/kg (40 µg SF6/kg) correspond to approx. 10 and 30 times, resp. the expected human imaging dose.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)

ADMINSTRATION
- Preparation of the animals: The rabbit was first anesthetized using pentobarbital (20 mg/kg, iv, auricular vein), then the neck, thoracic cage, and the lower abdomen were shaved. The animal was deeply anesthetized using a second intravenous injection of pentobarbital (20 mg/kg, auricular vein) and an intraperitoneal injection of urethane (1.4 g/kg). The jugular vein (for the injection of BR1) and the carotid artery (for blood sampling) were then catheterized and a tracheotomy was performed for artificial respiration and collection of exhaled air. Once the trachea had been canulated, the rabbit was paralyzed using an intramuscular injection of gallamine (10 mg/ kg). The rabbit's respiration was maintained throughout the experiment by connecting the tracheal catheter to an artificial respirator (connected to a pulmonary monitoring system) at a rate of approximately 20 respirations/min and at volume of 15 ml/respiration (corresponding to approximately 300 ml exhaled air/min). The penis was ligated to prevent the animal from urinating during the experiment.
- Dosing: The test or reference article was administered once by the intravenous route as a bolus injection via the jugular vein at a rate of about 0.2 ml/sec (Group A and B: injection 15 sec, Group C: injection 5 sec). The test or reference article was then flushed with saline to insure that all of the preparation had been administered.

SAMPLING
- Tissues and body fluids sampled: blood, exhaled air and urine.
* Blood: Blood samples (ca. 3 ml) were collected anaerobically in individually marked and tarred heparinized tubes. A blood sample was taken befeore the injection of the test or reference article and at other specified times (see any other information on materials section for additional information). The exact volume of the samples was determined after headspace analyses.
* Exhaled air: Throughout the experiment the number of respirations was recorded for each sampling period using a pulmonary monitoring system. In addition, before the commencement of each experimen, the average volume of exhaled air was determined for each respiration. Exhaled air samples were collected from the rabbit before the experiment and at the designated collection period (see Table 1 below) in empty uncontaminated Tedlar bags. Volumes of air taken within the first 8 min (between 150-600 ml) were introduced into 1 L bags, volumes taken between 8 and 20 min (between 900-1500 ml) into 3 L bags, and from 20 min to the end of the experiment (3000 ml) into 5 L bags. The exhaled air volume in each sample was determined by counting the number of resprirations during air sampling intervals and using the calibrated volume/respiration obtained before the experiment.
*Urine: at the end of the experiment (t = 120 min), urine from the bladder of each animal was collected anaerobically using tarred Vacutainers. The exact volume was determined after headspace analysis.
- Time and frequency of sampling: See Table 1 below.

ANALYTICS
- Methods for identification: The concentration SF6 was determined by using GC-TCD (test and reference articles) and GC-ECD (blood and urine samples and exhaled air samples).
- Detection limit: Quantification limit was evaluated to be the lowest concentration of the standards used in calibration, i.e. 4 µg SF6/ml-gas for GC-TCD and 0.015 ng SF6/ml-gas for GC-ECD. Detection limit was 0.5 pg SF6/ml-gas for GC-ECD.

Statistics:

The statistical procedure employed to compare the mean values of blood concentration of the reference article and the test article BR1 at dose of 1 ml/kg and 0.3 ml/kg was the repeated measures analysis of variance (ANOVA). The same method was used to compare the mean pulmonary eliminations. Comparisons between urinary elimination and total clearance of BR1 at dose of 1 ml/kg and 0.3 ml/kg were performed by means of t-tests.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:

SF6 disappears very rapidly from blood: about 80% of the injected dose is cleared after the first min following administration, and blood levels of SF6 drop to background levels by 11 min (dose: 0.3 ml/kg) and 20 min (dose: 1 ml/kg).

Details on excretion:

PULMONARY ELIMINATION
Approximately half of the injected SF6 was excreted in expired air during the first 30 seconds after administration and more than 75% after one minute.

URINE CONCENTRATIONS
Only ultra-trace quantities of SF6 (< 0.1 ng/g-urine or less than 0.001% of the injected dose) were detected in urine contained in the bladder at 2 h

Metabolite characterisation studies

Metabolites identified:

no

Any other information on results incl. tables

ABSORPTION:

Mean blood concentrations of SF6, measured after intravenous administration to the rabbit of the test article BR1 (dose levels = 0.3 and 1.0 ml/kg) and of the reference article are presented in Table 2. The results show that the blood level decay of SF6 is extremely fast. At 30 seconds after administration, the SF6 blood level had already decreased by more than two orders of magnitude, compared to the initial value C(0). SF6 concentrations were significantly higher (Anova, p< 0.05) only within the first 2 minutes after BR1 administration for Group B and only within the first minute for group C when compared to background levels measured in Group A (Table 2 below). After 3 min, levels in treatment groups fell to background levels (Anova, p > 0.05), and after 11 minutes (dose: 0.3 ml/kg) and 20 minutes (dose: 1 ml/kg) SF6 concentration in blood was below the quantification limit (< 0.01 ng/ml-blood). The extremely low levels of SF6 observed in blood within the first min after administration of BR1 show that half-life elimination of the gas is less than 1 min. The pharmacokinetic parameters are dose dependent at the dose levels tested. Mean values of the area under the blood-concentration-versus-time curve (AUC 0 to 120 min) were determined as 56 and 167 (ng/ml).min for respective dose levels of 0.3 ml/kg and 1.0 ml/kg. The proportionality of the AUCs to their respective dose levels, i.e. 186 (dose: 0.3 ml/kg) and 167 (dose: 1.0 ml/kg) shows that the systemic clearance is constant. Total clearance of SF6 was extremely rapid with a significant difference (t-test, p < 0.0005) between values of 218 and 231 ml/min/kg, at respective doses of 0.3 and 1.0 ml/kg.

Table 2: Blood concentrations of SF6 after intravenous administration of BR1 to the rabbit. Expressed asng SF6 ml-blood and as^o/_ooID of SF6/ml blood

	Control Group A Reference article		Group B BR1 (1 ml/kg)			Group C BR1 (0.3 ml/kg)
	Blood concentrations of SF6 after intravenous administration of SonoVue^TMto the rabbit
Time (min)	ng SF6/ml blood n = 5	SD	ng SF6/ml blood n = 6	SD	Expressed as^o/ooID of SF6 ml-blood**	ng SF6/ml blood n = 6	SD	Expressed as^o/ooID of SF6 ml-blood**
0.5 1 2 3 4 6 8 11 20 60 120	<0.01 nd nq nq nq nq nq nq <0.01 <0.01 <0.01	-- -- -- -- -- -- -- -- -- -- --	2.0* 0.7* 0.4* 0.17 0.10 0.06 0.04 0.03 0.01 <0.01 <0.01	0.8 0.3 0.2 0.08 0.05 0.03 0.03 0.02 0.02 0.003 0.003	0.016 0.006 0.003 0.001 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001	1.0* 0.5*° 0.1 0.10 0.06 0.03 0.015 < 0.01 <0.01 nq nq	0.3 0.4 0.3 0.04 0.03 0.01 0.005 -- -- -- --	0.024* 0.013 0.004 0.003 0.001 < 0.001 < 0.001 < 0.001 < 0.001 nq nq

nd=not detected

nq = not quantifiable i.e. all values<0.01 ng SF6/ml-headspace

<0.01 ng SF6/ml-blood: one or more animals in the group have not quantifiable values.

*significantly different ( p<0.05) versus control GroupA.

**Results expressed as^o/_ooof the injected dose (ID)/ml blood.

°GroupBversus Group C:significantly different ( p<0.05)

PULMONARY ELIMINATION Mean values of pulmonary elimination are presented in Table 3 below. SF6 is rapidly eliminated by the pulmonary route. Approximately half of the injected SF6 was excreted in expired air during the first 30 seconds after administration and more than 75% after one minute. Quantities of SF6 eliminated were significantly higher (Anova, p < 0.05) within the first 3 minutes after administration (Group B) and within the first 2 minutes (Group C) when compared with the background levels observed in the Control Group A. Cumulative results expressed as % of the injected dose show that SF6 was almost exclusively eliminated via the lungs in exhaled air. The cumulative percentage of the dose of SF6 eliminated in exhaled air exceeds 110% ID by 55 min for both groups. The high recovery can be attributed to the quantitative difficulties involved in measuring SF6. The results clearly show that the bulk of all SF6 injected is eliminated within minutes after administration, and nothing more than undetectable quantities ( < 0.01 ng) could possibly remain in the animal 2 h after administration.

Table 3: Pulmonary Elimination of SF6 after intravenous administration of BR1 to the rabbit. Expressed as µg SF6 and as % ID of the injected dose.

	Control Group A Reference article		Group B BR1 (1 ml/kg)			Group C BR1 (0.3 ml/kg)
	Elimination of SF6 (µg)
Time (min)	Mean n = 5	SD	Mean n = 6	SD	Cumulative elimination (%ID)++	Mean n = 6	SD	Cumulative elimination (%ID)++
0.5 1 2 3 4 6 8 11 15 20 30 55 90 120	0.01 0.01 nq nq nq nq nq nq nq nq nq nq nq nq	0 0.005 -- -- -- -- -- -- -- -- -- -- -- --	55.1* 52.5* 14.2* 4.3* 1.8 1.7 1.0 0.8 0.7 0.6 1.1 2.4+ 1.2+ 0.5+	6.9 7.8 3.3 1.6 0.8 0.6 0.3 0.2 0.2 0.2 0.6 0.6 0.3 0.1	44 87 98 102 103 105 105 106 107 107 108 110+ 111+ 111+	22.2*° 12.2*° 4.3*° 1.6 0.8 0.7 0.4 0.4 0.3 0.3 0.5 0.9+ 0.4+ 0.2+	3.7 3.6 0.7 0.4 0.3 0.1 0.1 0.3 0.1 0.1 0.2 0.3 0.1 0.1	56 87 98 102 104 106 107 107 108 109 110 112+ 113+ 114+

+ Extrapolated from elimination rates obtained during intervals from 45 to 55 min, 80 to 90 min and 110 to 120 min

++Results expressed as cumulative % of the injected dose (%ID) of SF6 eliminated

* Group B, Group C versus control Group A: significantly different (p <0.05)

° Group B versus Group C: significantly different (p <0.05)

URINE CONCENTRATIONS Mean values of SF6 concentrations in collected urine for the 2 h period after administration are presented in Table 4 below. The SF6 levels in urine were extremely low, hardly detectable. In Groups B and C, the urine levels were higher than background levels measured in Control Group A, but only 2 to 4 times higher than the quantitation limit (< 0.01 ng SF6/g-urine) or less than 0.001% ID (considering the largest volume of urine present in the rabbit bladder weighed only 20 g). These levels (0.2 ng in urine) are negligible compared to theµg quantities injected. Thus, it can be concluded that the renal pathway is not an elimination route for SF6.

Table 4: Urinary concentrations of SF6 2h after intravenous administration of BR1 to the rabbit. Expressed as ng SF6/g-urine and as %ID/g-urine

	Control Group A Reference article		Group B BR1 (1 ml/kg)		Group C BR1 (0.3 ml/kg)
	SF6 Concentration in Urine
Time: 120 min	Mean n = 5	SD	Mean n = 6	SD	Mean n = 6	SD
Ng SF6/g-urine %ID/g-urine	nq* --	-- --	0.04 0.00003	0.02 0.00001	0.02+ 0.00004	0.01 0.0003

nq*=not detected to not quantifiable (< 0.01 ng SF6/mLheadspace)

+Group C versus Group B: significantly different (p <0.0005)

Applicant's summary and conclusion

Conclusions:

Results of a blood kinetics study in rabbits demonstrate that the inert gas, SF6, administered by intravenous injection is rapidly eliminated via the pulmonary route and does not accumulate in the rabbit.

Executive summary:

SonoVue™ is an aqueous suspension of stabilized sulfur hexafluoride (SF6) bubbles used as a contrast agent for echography. The aim of this study was to establish the blood kinetics, pulmonary elimination, and possible renal elimination of SF6 after a single intravenous injection of SonoVue™ to the rabbit.

SonoVue™ was administered at two dose levels, 0.3 ml/kg (12 µg SF6/kg) and 1.0 ml/kg (40 µg SF6/ kg), corresponding to approximately 10 and 30 times, respectively, the expected human imaging dose. SF6 in blood, urine, and exhaled air was assayed using GC.

Results show that after administration of SonoVue™, SF6 disappears very rapidly from the blood: about 80% of the injected dose is cleared after the first minute following administration of SonoVue™, and blood levels of SF6 drop to trace levels 6 min after administration (<0.1 ng/ml blood). Total clearance of SF6 from blood is extremely rapid (218 to 231 ml/min/kg), and half-life elimination of blood SF6 is less than 1 minute. SF6 is eliminated almost exclusively via the pulmonary route following administration of SonoVue™. More than 90% of the injected dose recovered is eliminated within 3 min. Only ultra-trace quantities of SF6 (< 0.1 ng/g urine or less than 0.001% of the injected dose) were detected in urine contained in the bladder at 2 h, thus showing that renal elimination is not an elimination route for SF6. Results of this study demonstrate that the inert gas SF6, administered by intravenous injection is rapidly eliminated via the pulmonary route and does not accumulate in the rabbit.