Tetradecafluorohexane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 5-20, 1997

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:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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suspension must be withdrawn from the vial with the supplied vented 5 µm filter dispensing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

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

young adult male and female mice, Crl:CD-1 (ICR) BR strain. Standard procedures were followed for housing, handling, feeding and care of the animals. Alter acclimation for at least 7 days, animals were randomly assigned into 5 groups (6 animals/sex/group) and received designed treatments.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were 8 weeks old with body weights of 29.2-34.1 (males) and 22.2-28.6 (females) at initiation of treatment.

Administration / exposure

Route of administration:

intravenous

Vehicle:

Water

Details on exposure:

AF0150 and Positive Control Preparations: AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/ml and was used within 30 minutes alter reconstitution.

Animals received IV injection of a single dose of AF0150 or saline (negative control), and oral administration of 80 mg/kg cyclophosphamide (positive control).
Table 1. Micronucleus Studv Desisn in Mice


Treatment Dosing Vol. Route
(mUkg) of Dosing Harvest Time, 24 hr Harvest Time, 48 hr

Male Female Male Female
200 mg/kg AF0150 5.0 IV 6 6 - -
400 mg/kg AF0150 10 IV 6 6 6 6
800 mg/kg AF0150 20 IV 6 6 6 6
Saline 20 IV 6 6 6 - -
Cyclophosphamide 10 PO 6 6 - -

Duration of treatment / exposure:

Animals received IV injection of a single dose of AF0150 or saline (negative control), and oral administration of 80 mg/kg cyclophosphamide (positive control).

Frequency of treatment:

unique administration

Post exposure period:

24-48h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw (total dose)

Doses / concentrations 4

Dose / conc.:

800 mg/kg bw (total dose)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

80 mg/kg oral cyclophosphamide

Examinations

Tissues and cell types examined:

Toxic signs were observed at least daily for all animals throughout study

Details of tissue and slide preparation:

Bone marrow was collected from the hind limb and transferred to centrifuge tubes containing 3-5 ml bovine serum. The cells were centrifuged, prepared for slides, fixed in methanol and stained with May-Grunwald and Giemsa.

Evaluation criteria:

The slides were scored for polychromatic erythrocyte (PCE), normochromatic erythrocyte (NCE) and micronucleated PCE. The micronucleus frequency, expressed as percentage of micronuleated cells, was determined by analyzing the number of micronucleated PCEs from 2000 PCEs per animal.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Treatments with AF0150, saline or CP had no significant toxic signs in all animals. As seen in Table 2, AF0150 at all doses was no cytotoxic to bone marrow in ternis of PCE:NCE ratio. There was no significant difference in micronucleated PCEs between AF0150- and saline-treated mice. The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs in bath sexes as compared to saline control group.

Any other information on results incl. tables

Table 2.Micronucleus Assay Summary

 

TREATMENT	DOSE	HARVEST TIME (HA)	% MICRONUCLEATED PCEs                                                                            RATIO PCE:NCE MEAN OF 2000 PER ANIMAL * S.E.                                               MEAN s S.E. MALES               FEMALES           TOTAL             MALES                FEMALES
controls
VEHICLE	0.0% saline	24 hr	0 08 4 0.03	0.04 s 0.03	0.06 ± 0.02	0.59 s 0.04	0.79 ± 0.05
		48 tu	0.08 ± 0.03	0.04 s 0.03	0.06 .± 0.02	0.46 s 0.02	0.52 ± 0.06
POSTIVE	CP 80.0 mg/kg	24 hr	3.52 ± 0 19»	2.28 * 0.29*	2.90 ± 0.26•	0.71 4 0.07	0.68 s 0.09
TEST ARTICLE	200 mg/ka	24 hr	0-05 ± 0.03	0.07 4 0.01	0.06 s 0.02	0.74 * 0.09	0.76 * 0.05
	400 mg/kg	24 hr	0.15 ± 0 04	0.08 4. 0.04	0,12 ± 0.03	0,69 * 0.04	0.52 4 0.04
	800meg	24 hr	0.05 ± 0.02	0.10 4 0.04	0.08 s 0.02	0.64 * 0.06	0.72 ± 0.11
		48 hr	0.03 * 0.01	0.04 ± 0.03	0.04 s 0.02	0 50 * 0.08	0.43 ± 0.02

 

· Significantly greater than the corresponding vehicle control, p<0.01.

CP = Cvclophosphamide

PCE =Polychromatic erythrocyte NCE = Normochromatic erythrocyte

Applicant's summary and conclusion

Conclusions:

AF0150, which active ingredient is tetradecafluorohexane, is not toxic to bone marrow in mice.

Executive summary:

Treatments with AF0150, saline or CP had no significant toxic signs in all animals. As seen in Table 2, AF0150 at all doses was no cytotoxic to bone marrow in ternis of PCE:NCE ratio. There was no significant difference in micronucleated PCEs between AF0150- and saline-treated mice. The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs in bath sexes as compared to saline control group.