Tetradecafluorohexane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 29 — October 11, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

No study was available assessing short-term repeated dose toxicity of tetradecafluorohexane by oral route.
However, extensive data was available for the drug AF0150, which new drug application (NDA) was successfully assessed by US-FDA. AF0150 is tetadecafluorohexane in phospholipids, administered by intraveinous route. As discussed in toxicokinetics statement, formulation and route of administration of AF0150 is assumed to dramatically increase bioavalability of tetradecafluorohexane, without modifying further ADME since tetradecafluorohexane is not metabolized and rapidly eliminated in expired air.
IV AF0150 data was therefore considered relevant to assess short-term repeated dose toxicity of tetradecafluorohexane by oral route.

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:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

IV administration

GLP compliance:

yes

Remarks:

US-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:

CD (SD) BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

male and female rats (95 each), Crl:CD (SD) BR VAF/Plus.
The animais were 39-45 days old and weighed 151.7-189.1 g
(males) and 149.4-199.0 g (females) at initiation of treatment. Standard procedures were followed for housing, handling, feeding and care of the animais. The rats were acclimated for 8 days before initiation of treatment and, alter exclusion of those with body weight exceeding ±2SD), randomly assigned into 4 groups (20/sex/group), as seen in table 1.

Administration / exposure

Route of administration:

other: Intraveinous

Details on route of administration:

Bolus

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

29 daily IV injection in rats followed by a 14-day recovery

Frequency of treatment:

Daily IV Bolus

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

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. Animais received 0 (saline), 50, 200, or 400 mg/kg AF0150 through IV injection. Individual doses were calculated based on the most recent recorded body weight. Dose groups are shown in the Table 1.

Table 1. Re eated Dose Toxicity Study in Rats
Group AF0150 Dose*
(mg/kg/day) IV Volume
(ml/kg/day) Number of Rats**
Male Female
1(Control) # 0 10 20 20
2(Low) 50 1.25 20 20
3(Mid) 200 5 20 20
4(High) 400 10 20 20

* AF0150 was reconstituted in SWFI to final concentration of 40 mg/ml.
** 5 rats/sex/group were sacrificed on day 17; 10 rats/sex/group were sacrificed on day 29 or 30; 5 rats/sex/group were treated daily for 29 days and then sacrificed after 15-day recovery period.
# Received 0.9% NaC1 for Injection (10 ml/kg).

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Observation Parameters: toxicity signs were observed daily. Body weigh and food consumption were recorded weekly. Ophthalmic examination was performed before initiation of treatment and during week 4 of treatment. Blood and urine samples were collected on Days 18 and 44 (5 rats/sex/group) and on Days 30 or 31 (10 rats/sex/group) for hematology, blood chemistry and urinalysis.

Sacrifice and pathology:

At each scheduled sacrifice (as indicated in Table 1), animais were necropsied for macroscopic examination including measurement of organ weights and histopathology.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

on Day 30/31, the following changes were observed and were statistically significant: lower blood creatinine in male rats for all doses; lower blood total protein for male and lower globulin for males and females at the doses of 200 and 400 mg/kg/day; lower aspartate aminotransferase for males given 400 mg/kg/day and females at all dose levels; lower alanine aminotransferase for males and females given 200 or 400 mg/kg/day, and lower alkaline phosphatase for males at all dose levels. Alter a 15-day recovery period, alanine aminotransferase remained decreased in females given 200 and 400 mg/kg/day. On Day 18, statistically significant decreases in blood urea nitrogen and alanine aminotransferase were shown in females given 200 and 400 mg/kg/day.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

there was no significant organ weight changes on Day 18. But on Day 30/31, liver and spleen weights significantly increased in female rats given 200 and 400 mg/kg/day and the spleen weight continued to increase at the end of 15-day recovery period in rats treated with 400 mg/kg/day. Although the weights of the liver and spleen were not significantly increased on day 30-31 in the male rats given AF0150, there appeared to be a trend toward increased spleen weight.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1. Vacuolated macrophages were observed in the followed organs on Days 18 and 30/31 without significant reduction after the 14-day recovery period.
spleen and mesenteric lymph node of animais (both sexes) given the AF0150 at all dose
levels with the higher incidence and severity at the dose of 400 mg/kg/day;
Liver Kupffer cells in some animais (both sexes) given 200 mg/kg/day and most animais
given 400 mg/kg/day;
Thymus, bronchial-associated lymphoid tissue (lung), adrenal medulla and glomeruli of some
animais given 400 mg/kg/day;
Uterus and cervix of females given 200 and 400 mg/kg/day;
Lymph nodes in mammary gland;
Negative vacuolation in bone marrow cells;
2. Eosinophil infiltrates in mesenteric lymph nodes and perivascular eosinophil infiltrate in the lungs increased in males and females given 200 and 400 mg/kg/day on Days 18. On Day 30/31, eosinophil infiltrate was shown only in mesenteric lymph nodes. These changes decreased after the recovery period. NOAEL was 50 mg/kg/day (HED: 8 mg/kg/day; HDM: 65-fold)
3. Increase in extramedullary hematopoiesis in the spleen was observed with a dose-dependence in males, and at 400 mg/kg/day in females on Day 18. Only males showed increased extramedullary hematopoiesis at 200 or 400 mg/kg/day. These change slightly increased after recovery period.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

food consumption and compound intake

haematology

mortality

ophthalmological examination

urinalysis

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

3

Dose descriptor:

LOAEL

Effect level:

ca. 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

4

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

2

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Clinical Signs: allanimals survived to scheduled termination. There were no remarkable toxic signs associated with AF0150 treatment.

Body Weight and Food Consumption: AF0150 treatment had no significant effects on body weight and food consumption in any of the animals.

Ophthalmology:no remarkable ophthalmic changes were observed pre- and week 4 post­AF0150 treatment.

Clinical Chemistry: on Day 30/31, the following changes were observed and were statistically significant: lower blood creatinine in male rats for all doses; lower blood total protein for male and lower globulin for males and females at the doses of 200 and 400 mg/kg/day; lower aspartate aminotransferase for males given 400 mg/kg/day and females at all dose levels; lower alanine aminotransferase for males and females given 200 or 400 mg/kg/day, and lower alkaline phosphatase for males at all dose levels. Alter a 15-day recovery period, alanine aminotransferase remained decreased in females given 200 and 400 mg/kg/day. On Day 18, statistically significan

decreases in blood urea nitrogen and alanine aminotransferase were shown in females given 200 and 400 mg/kg/day.

Hematology and Urinalysis:Hematology and urinalysis were not remarkable.

Organ Weight:there was no significant organ weight changes on Day 18. But on Day 30/31, liver and spleen weights significantly increased in female rats given 200 and 400 mg/kg/day and the spleen weight continued to increase at the end of 15-day recovery period in rats treated with 400 mg/kg/day. Although the weights of the liver and spleen were not significantly increased on day 30-31 in the male rats given AF0150, there appeared to be a trend toward increased spleen weight.

Histopathology:

1.     Vacuolated macrophages were observed in the followed organs on Days 18 and 30/31 without significant reduction after the 14-day recovery period.

spleen and mesenteric lymph node of animals (both sexes) given the AF0150 at all dose levels with the higher incidence and severity at the dose of 400 mg/kg/day;

Liver Kupffer cells in some animals (both sexes) given 200 mg/kg/day and most animals given 400 mg/kg/day;

Thymus, bronchial-associated lymphoid tissue (lung), adrenal medulla and glomeruli of some animals given 400 mg/kg/day;

Uterus and cervix of females given 200 and 400 mg/kg/day;

Lymph nodes in mammary gland;

Negative vacuolation in bone marrow cells;

2.     Eosinophil infiltrates in mesenteric lymph nodes and perivascular eosinophil infiltrate in the lungs increased in males and females given 200 and 400 mg/kg/day on Days 18. On Day 30/31, eosinophil infiltrate was shown only in mesenteric lymph nodes. These changes decreased after the recovery period. NOAELwas 50 mg/kg/day.

3.     Increase in extramedullary hematopoiesis in the spleen was observed with a dose-dependence in males, and at 400 mg/kg/day in females on Day 18. Only males showed increased extramedullary hematopoiesis at 200 or 400 mg/kg/day. These change slightly increased after recovery period.

Applicant's summary and conclusion

Conclusions:

IV NOAEL: 50 mg/kg/d. As oral absorption potential is estimated to be 10%, Oral NOAEL is estimated to be 500 mg/kg/d
Vacuolisation of macrophages observed at all dose levels, with unknown clinical significance. Vacuolisation may only be relevant for AF0150 particular galenic form, i.e. phospholipid microspheres, and therfore not be relevant for tetradecafluorohexane.

Executive summary:

1. AF0150 at all dose levels induced macrophage vacuolation in multiple tissues in a dose­dependent manner on Day 30 and at the end of a 15-day recovery period. The organs/tissues rich in monocytes/macrophages showed the highest incidence of vacuolated macrophages, such as in spleen and lymph nodes, but not in bone marrow. Eosinophil infiltrates in

mesenteric lymph nodes and perivascular eosinophil infiltrate in the lungs increased in males and females, and these changes decreased after the recovery period. Increase in extramedullary hematopoiesis in the spleen was observed at 200 or 400 mg/kg/day. These changes slightly increased after the recovery period.NOAELfor both effects was 50 mg/kg/day.

2. It is not clear if the source of vacuolation macrophages in tissues was from vacuolated circulating monocytes. if the macrophages in tissues take up the microbubbles, how the microbubbles cross the endothelium.

3.     The nature of the vacuole in the macrophages was not described. It is unclear if the vacuole represent phagocytosed microbubbles.

4.     The fate of the vacuolated macrophages and long-term effects of the vacuolation on macrophage function needs to be addressed, such as phagocytosis, cytokine production, antigen presentation; and host resistance to bacterial challenge (macrophage/monocytes­mediated defense)

5.     Most blood chemistry parameters with AF0150-related changes were associated with liver function. Although increases rather than decreases in these parameters are generally related to organ acute toxicity (increased or destroyed cell membrane permeability), chronic toxicity to the liver may decrease liver function (without significantly affecting the cell member intact) and thus lower liver enzyme markers in the blood. Increase in the liver weight could be supportive of a chronic injury process.