Tetradecafluorohexane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 28-November 21, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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suspension must be withdrawn from the vial with the supplied vented 5 µm filter dispensing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.

Specific details on test material used for the study:

AF0150 Lot number: ZZ15031 (400 mg/vial)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary Test (Dose Range-Finding): TA98, TA100 and Wp2uvrA were incubated with AF0150 at the dose range from 0.4 to 20 mg/plate (total 8 doses) in the absence and presence of S9 activation. No cytotoxicity (decreased number of revertant colonies, or thinning/disppearance of the bacterial background lawn) was observed at all dose levels. The maximal dose, 20 mg/plate, was selected for mutagenicity assay.

Vehicle / solvent:

Water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene; ICR-191

Details on test system and experimental conditions:

Preliminary Test (Dose Range-Finding): TA98, TA100 and Wp2uvrA were incubated with AF0150 at the dose range from 0.4 to 20 mg/plate (total 8 doses) in the absence and presence of S9 activation. No cytotoxicity (decreased number of revertant colonies, or thinning/disppearance of the bacterial background lawn) was observed at all dose levels. The maximal dose, 20 mg/plate, was selected for mutagenicity assay.
AF0150 and Positive Control Preparations: AF0150 (400 mg fill) was reconstituted with 10 ml sterile deionized water to a final concentration of 40 mg/ml. For low dose groups (0.4-2 mg/plate), 4 mg/ml AF0150 stock solution was made by dilution of 40 mg/ml. All AF0150 stock solutions were used within 30 minutes alter reconstitution. Positive control chemicals included 2- aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide [Preparation methods were not indicated].
Mutagenicity Assay: The five bacterial strains were pre-incubated in culture tubes (tightly capped) with AF0150 at the doses of 1-20 mg per plate in the absence and presence of rat liver S9 for 2 hours at 37°C. The overlay (top) agar (selection agar medium) containing histidine/biotin or tryptophan was then added to the tubes. The agar and preincubation mixtures were overlaid onto petri dishes containing minimal bottom agar, in triplicate for all dose levels and controls (negative and positive). Following incubation of the petri dishes for 48 hours at 37°C, the number of revenant colonies on each petri dish was counted manually (for AF0150 and negative control plates) or automatically (with automated colony counter for positive control plates).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

AF0150 at the doses of 1-20 mg/plate had no significant cytotoxicity and did not increase revenants in all 5 bacterial strains covered both C-C and A-T point mutations in the absence and presence of a rat liver S9. Positive control compounds (2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide) significantly increased reverse mutation in all bacterial strains.

In addition, bubbles were observed in the top agar at doses of 4 mg AF0150 per plate and above in both the presence and absence of S9 mix in dose range finding study.

Table 1.Mitotic Index (MI, %) and Chromosomal Aberration in PCE (CA-PCE, %)
of AF0150-Treated Human LvmnhocvteIn vitro

Treatment	Initial Assay				Confirmatory Assay
	-S9		+S9		-S9		+S9
	MI	CA-PCE	MI	CA-PCE	MI	CA-PCE	MI	CA-PCE
2 mg/mi *	5.4	0.5	4.1	0	2.6	0.5	5.4	0.5
3 mg/m1	4.5	1.0	3.9	0	2.8	0	2.9	1.0
4 mg/mi	6.5	0	4.8	0.5	2.6	0.5	4.2	1.0
5 mg/m1	3.4	0	3.3	1.0	2.9	0	4.1	0
CPt			1.6	32			3.6	12
MMCt	0.8	62			1.8	34
H2O	6.4	1.5	4.5	0	3.5	0	4.2	0

 

* AF0150 concentration in cultured human lymphocytes.

tCP (cyclophosphamide, with S9 activation) and MMC (mitomycin C, without S9 activation) were positive controls.

Applicant's summary and conclusion

Conclusions:

AF0150, which active ingredient is tetradecafluorohexane, is not mutagenic.

Executive summary:

AF150 at the dose of 1-20 mg/plate did not result in significant increase in bacterial reverse mutation. However, there was also no evidence of significant cytotoxicity at the highest dose, 20 mg/plate in terms of changes on revertant numbers or bacterial background lawn. This suggests that the dose selection may not be sufficient.