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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1996
Report date:
1997

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
20 male guinea pigs were assigned into 4 groups (5 animais/group) and treated. Different dilutions of serum samples collected in ASA procedure were intradermally injected into each animal with 0.05 ml per injection site and at least 1 cm apart between sites. At approximately 4.5 hours following serum injection, the animais in Groups 6, 7, and 8 received 1 ml/kg of Evan's Blue/AF0150 mixture (0.1% Evan's Blue and 20 mg/ml AF0150) by IV injection, and the animais in Group 9 received 0.5 ml/animal of Evan's Blue/BSA mixture (0.1% Evan's Blue and 10 mg/mi BSA). Approximately 30 minutes after IV injection, the diameter of blue dye staining area at each intradermal injection site was measured. Clinical signs, body weight and pathology were not observed.
GLP compliance:
yes
Type of study:
intracutaneous test
Justification for non-LLNA method:
Exeisting in vivo data relevant to provide information on sensitization potential of tetradecafluorohexane.

Test material

Constituent 1
Chemical structure
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.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Crl:(HA)BR
Sex:
not specified
Details on test animals and environmental conditions:
albino guinea pigs, Crl:(HA)BR strain, were 5-10 weeks old at initiation of treatment with
body weight of 470-645 g (for PCA). Standard procedures were followed for housing, handling, feeding and care of the animais. The animais were acclimated for 5 days before initiation of treatment.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal
Vehicle:
water
Concentration / amount:
20 mg/mL
Day(s)/duration:
33
Adequacy of induction:
not specified
Challenge
No.:
#1
Route:
intradermal
Vehicle:
water
Concentration / amount:
20 mg/kg
Day(s)/duration:
5h
Adequacy of challenge:
not specified
No. of animals per dose:
5
Details on study design:
AF0150 and BSA Preparation: AF0150 (200 mg fill vial) was reconstituted with 10 ml of sterile water to a concentration of 20 mg/ml and used within 30 minutes of preparation. 10 mg/ml of BSA was prepared by adding saline to 0.1 g BSA to a total volume of 10 ml. Saline (0.9% NaC1) was used as negative control. For induction where applied, AF0150 was mixed with FCA (Freund's Complete Adjuvant) at the ratio of 1:1 (v/v).
Procedure for Passive Cutaneous Anaphylaxis (PCA) (Table 2): 20 male guinea pigs were assigned into 4 groups (5 animais/group) and treated as shown in Table 2. Different dilutions of serum samples collected in ASA procedure were intradermally injected into each animal with 0.05 ml per injection site and at least 1 cm apart between sites. At approximately 4.5 hours following serum injection, the animais in Groups 6, 7, and 8 received 1 ml/kg of Evan's Blue/AF0150 mixture (0.1% Evan's Blue and 20 mg/ml AF0150) by IV injection, and the animais in Group 9 received 0.5 ml/animal of Evan's Blue/BSA mixture (0.1% Evan's Blue and 10 mg/mi BSA). Approximately 30 minutes after IV injection, the diameter of blue dye staining area at each intradermal injection site was measured. Clinical signs, body weight and pathology were not observed.

The serums were collected from animais sensitized with AF0150 or BSA or saline in ASA study. PCA Reactions were measured based on the diameter of blue dye staining area at serum injection sites.
Challenge controls:
Group
,., Treatment Dilutions Administered
6 Group 1 serum 1:1, 1:10, 1:100, 1:1,000, and 1:10,000
7 Group 3 serum 1:1, 1:10, 1:100. 1:1,000, and 1:10,000
8 Group 4 serum 1:1, 1:10, 1:100, 1:1,000, and 1:10,000
9 Group 5 serum 1:100, 1:1,000, and 1:10,000

Positive control substance(s):
yes
Remarks:
BSA

Results and discussion

Positive control results:
the serum prepared from BSA-induced animais resulted in a positive PCA reactions at 9 of the 10 injection sites with 1:100 serum dilutions and at one site with 1:1000 serum dilution.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
other: area of blue dye staining at the injection sites
Hours after challenge:
5
Group:
negative control
Dose level:
0 mg/kg
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No measurable area of blue dye staining at the injection sites
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: area of blue dye staining at the injection sites
Hours after challenge:
5
Group:
test chemical
Dose level:
20mg/kg
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No measurable area of blue dye staining at the injection sites
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
5
Group:
positive control
Dose level:
1:100
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
positive PCA reactions
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
5
Group:
positive control
Dose level:
1:1000
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
positive PCA reactions
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
other: No antigenicity
Executive summary:

No systemic or interdermal antigenicity was observed after a 33d induction phase.