Tetradecafluorohexane

Acute Toxicity: other routes

Administrative data

Endpoint:

acute toxicity: other routes

Remarks:

intraveinous

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase: August 14-28, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

An acute toxicity study by oral route was performed in the context of REACh registration of tetradecafluorohexane.
Extensive data on tetradecafluorohexane is available for the IV route in the context of the new drug application 21-191 for tetradecafluorohexane in phospholipid microsphere. As some litterature data reports exposure of tetradecafluorohexane by inhalation without noticeable effects on animals and humans, generation of new data to assess acute toxicity by other routes was conssidered in contradiction with 3R principles.

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: US-FDA - Guidance for Industry - Single Dose Acute Toxicity Testing for Pharmaceuticals

Deviations:

not specified

Principles of method if other than guideline:

AF0150 Preparation and Administration: AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/kg and used within 30 minutes. Animals received 0 (saline), 50, 200, or 400 mg/kg AF0150 through an IV injection into a lateral tail vein. Individual doses were caiculated based on the day 1 body weight. Dose groups are shown in the Table 1.
Observation Parameters: mortality and toxicity signs were observed twice daily (AM and PM) for 14 days. On the day 1, animals were examined predose and at 5 minutes, 0,5, 1, 2, 3 hours postdose for toxicity signs. Body weight and food consumption were recorded weekly. Blood samples were taken from the jugular vein on Days 2 and 8 (5 rats/sex/group), on Day 4 (10 rats/sex/group), and on Day 15 (ail remaining rats) for hematology and clinical chemistry analysis. The necropsy was performed from 5 rats/sex/group on Days 2 and 8 and remaining rats on Day 15, including organ weights, macroscopic and histopathological examination (the selected organs/tissues were listed in Table 1 in the Toxicology Summary on page 141).
Table 1. Ex anded Acute Toxicity Studv in Rats
Group AF0150 Dose*
(mg/kg) Number of Rats** IV Volume
(ml/kg)
Male Female
1(Control) e 0 20 20 10
2(Low) 50 20 20 1.25
3(Mid) - 200 20 20 5
4(High) 400 20 20 10

* AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.
** Sacrificed 5 rats/sex/group on Days 2 and 8, and 10 rats/sex/group on Day 15. Received 0.9% NaC1 for Injection

GLP compliance:

yes

Remarks:

FDA audited

Test material

Test material information

Constituent 1

Reference substance name:

Tetradecafluorohexane

EC Number:

206-585-0

EC Name:

Tetradecafluorohexane

Cas Number:

355-42-0

Molecular formula:

C6F14

IUPAC Name:

tetradecafluorohexane

Test material form:

liquid

Remarks:

Preparation for iv injection

Details on test material:

Imagent® Kit for the preparation of perflexane lipid microspheres for injectable suspension,
is a sterile, non-pyrogenic white powder with a diluted perflexane headspace that, after
reconstitution into a suspension of microspheres, is used for contrast enhancement during the
indicated ultrasound imaging procedures.
The contents of the 200 mg Imagent powder vial are sterile and non-pyrogenic. Each vial
of Imagent® powder contains 9.2 mg 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
75 mg hydroxyethyl starch; 2.1 mg poloxamer 188; 75 mg sodium chloride; and 36 mg
sodium phosphate buffer in a vial filled with a mixture of 17% v/v perflexane vapor in
nitrogen.
After reconstitution with 10 mL of the provided Sterile Water for Injection, USP, the
contents of the vial yield an opaque white suspension for injection. The reconstituted
suspension must be withdrawn from the vial with the supplied vented 5 µm filter dispensing
pin.
Each mL of reconstituted aqueous suspension contains a maximum of 13.7 x 108
microspheres, 92 µg perflexane, 0.92 mg DMPC; 7.5 mg hydroxyethyl starch; and 0.21 mg
poloxamer 188. The reconstituted product is iso-osmolar and has a pH between 6.7 to 7.7.
Table 1. Microsphere Size Distribution
DIAMETER
Mean Volume Weighted Median: 6 µm
Number per mL
Mean (% of Total)
ALL SIZES (Total) 5.9-13.7 x 108
(100%)
<3 µm 7 x 108
(78.8%)
3 - 10 µm 2 x 108
(21.0 %)
>10 µm 0.01 x 108
(0.2 %)
Upper limit 20 µm
The active moiety, the microsphere, comprises two critical components: perflexane, the
gaseous component, and DMPC, the lipid membrane component.
Perflexane is chemically characterized as n-perfluorohexane with a molecular weight of 338
atomic mass units and an empirical formula of C6F14. Perflexane has the following structural
formula:
FF FF F
F
F
F F F
F
F
F F
DMPC is a semi-synthetic (not of animal origin) phospholipid and is chemically
characterized as 1, 2,-dimyristoyl-sn-glycero-3-phosphocholine with a molecular weight of
678 atomic mass units and an empirical formula of C36H72NO8P. DMPC has the following
structural formula:
O H C
O
O CH2
H2C
O
O
P
O O
O
N(CH3)3 -
+
Imagent Kit for the Preparation of Perflexane-Lipid Microspheres Injectable Suspension is
supplied for single-use and each kit contains a 10-mL glass vial containing 200 mg of
Imagent powder, a 20-mL plastic vial of Sterile Water for Injection, a 10-mL disposable
plastic sterile syringe, a sterile, vented 5 µm filter dispensing pin, and a package insert.
The powder vial must be reconstituted with 10 mL supplied Sterile Water for Injection and
then withdrawn from the vial with the provided vented 5 µm filter dispensing pin as
described under DOSAGE AND ADMINISTRATION – Drug Handling and Preparation.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD (SD) BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

: male and female rats (90 each), Crl:CD (SD) BR VAF/Plus. The animals were 38-44 day-old and weighed 136.6-176.2 g
(males) and 108.8-153.4 g (females) at initiation of treatment. Standard procedures were followed for housing, handling, feeding and care of the animals. The rats were acclimated for 7 days before initiation of treatment. 80 rats from each sex (after exclusion of those with body weight exceeding ±2SD) were randomly assigned into 4 groups (20/sex/group), as seen in table1.

Administration / exposure

Route of administration:

intravenous

Vehicle:

water

Details on exposure:

AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/kg and used within 30 minutes. Animals received 0 (saline), 50, 200, or 400 mg/kg AF0150 through an IV injection into a lateral tail vein. Individual doses were caiculated based on the day 1 body weight. Dose groups are shown in the Table 1.

Doses:

Table 1. Ex anded Acute Toxicity Studv in Rats
Group AF0150 Dose*
(mg/kg) Number of Rats** IV Volume
(ml/kg)
Male Female
1(Control) e 0 20 20 10
2(Low) 50 20 20 1.25
3(Mid) - 200 20 20 5
4(High) 400 20 20 10

* AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.
** Sacrificed 5 rats/sex/group on Days 2 and 8, and 10 rats/sex/group on Day 15. Received 0.9% NaC1 for Injection

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

mortality and toxicity signs were observed twice daily (AM and PM) for 14 days. On the day 1, animals were examined predose and at 5 minutes, 0,5, 1, 2, 3 hours postdose for toxicity signs. Body weight and food consumption were recorded weekly. Blood samples were taken from the jugular vein on Days 2 and 8 (5 rats/sex/group), on Day 4 (10 rats/sex/group), and on Day 15 (ail remaining rats) for hematology and clinical chemistry analysis. The necropsy was performed from 5 rats/sex/group on Days 2 and 8 and remaining rats on Day 15, including organ weights, macroscopic and histopathological examination (the selected organs/tissues were listed in Table 1 in the Toxicology Summary on page 141).

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

>= 400 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

On Day 4, one male rat in the control group and 3 rats (1 male and 2 female) in 50 mg/kg AF0150 group, died following blood collection. The deaths were very likely related to the blood collection procedure but not to treatment. All other animals survived to the scheduled sacrifice.

Clinical signs:

no overt signs of toxicity were noted after an IV injection of AF0150 at doses of 50-400 mg/kg.

Body weight:

AF0150 treatment had no effects on body weight gain and food consumption.

Gross pathology:

there were no remarkable macroscopic changes associated with AF0150 treatment at all doses and time points. However, histopathologic observation showed that AF0150 induced infiltration of vacuolated macrophages in the spleen and mesenteric lymph nodes at all dose groups in a dose-dependent and time-dependent manner. The no observed effect level (NOEL) could not be estimated since the vacuolated macrophages were found in rats given 50 mg/kg, the lowest dose group in this study.

Other findings:

Hematology and Clinical Chemistry: slight decreases in platelets, neutrophils, RBC, Hb, HCT, serum total protein and albumin, and slight increases in BUN, serum creatinine and inorganic phosphorous were reported in some of AF0150-treated rats. However, the changes in these parameters were independent of AF0150 doses, sex of animals and time post dosing, suggesting these findings were probably not related to AF0150 treatment.
Organ Weight: no significant change in the absolute weights and relative weights (organ-to-body weight and organ-to-brain) was found in most organs, including lung, heart and brain. The following slight changes were seen in AF0150-treated male rats on Day 15: decrease in the absolute weights and relative weights (organ-to-brain) of right adrenal gland, left kidney at the dose of 400mg/kg and increase in the absolute weight of liver at the dose of 50 mg/kg. These changes seem not to be related to AF0150 dose, sex and/or contralateral organs and thus may not be associated with AF0150 treatment

Any other information on results incl. tables

Table 1.Ex anded Acute Toxicity Studv in Rats

Group	AF0150 Dose* (mg/kg)	Number of Rats**		IV Volume (ml/kg)
		Male	Female
1(Control)^e	0	20	20	10
2(Low)	50	20	20	1.25
3(Mid)	- 200	20	20	5
4(High)	400	20	20	10

 

* AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.

** Sacrificed 5 rats/sex/group on Days 2 and 8, and 10 rats/sex/group on Day 15. Received 0.9% NaC1 for Injection

Results

Applicant's summary and conclusion

Conclusions:

IV injection of AF0150 at the doses of 50-400 mg/kg did not result in significant acute toxicity in rats.

On the basis of the toxicokinetics report estimating 10% oral absorption, acute oral toxicity in rat, if relevant, is not expected to be lower than 4000 mg/kg.

Executive summary:

IV injection of AF0150 at the doses of 50-400 mg/kg did not result in significant acute toxicity in rats. However, histopathologic examination showed that AF0150 induced macrophage vacuolation in the spleen and mesenteric lymph nodes at all dose groups in a dose-dependent and time-dependent manner. The cause of vacuolisation and fate of the vacuolated macrophages, particularly potential effects on macrophage function, needs to be further addressed. Vacuolisation observed may be phagocyted phospholipid microspheres, and therefore not be relevant for exposure to tetradecafluorohexane.

On the basis of the toxicokinetics report estimating 10% oral absorption, acute oral toxicity in rat, if relevant, is not expected to be lower than 4000 mg/kg.