Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
SEPTEMBER 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was carried out with a QA-statement according to a well-defined protocol. Furthermore, the stud y was assessed in the current risk assessment report under the excisting substance regulation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: SCCNFP in-vitro assessment of percutaneous absorption guidelines
Deviations:
not specified
Principles of method if other than guideline:
For the present human skin penetration study, 12 human epidermal membranes were clamped in dosage chambers. Permeation of HHCB was then measured over a 24 hrs period. The epidermal membranes were then tape stripped 10 times and the radiolabel content of the strips and remaining epidermis determined.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): HHCB
- Substance type: Fragrance material

Unlabeled material:
- Analytical purity: 98.5%
- Lot/batch No.: 7953-I
- Expiration date of the lot/batch: December 2001

Radiolabeled material:
- Radiochemical purity (if radiolabelling): 99.3%
- Specific activity (if radiolabelling): 36.7 mCi/mmol
- Locations of the label (if radiolabelling): Centered in the aromatic ring
- Other: The radiolabeled fragrance material was prepared by Wizard Laboratories, Inc., CA.
Radiolabelling:
yes

Administration / exposure

Type of coverage:
other: The skin samples were mounted in a exposure chamber with the stratum corneum facing the donor chamber.
Vehicle:
ethanol
Duration of exposure:
24 hrs
Doses:
- Nominal doses: 20 uL/cm2 target dose of a 1% solution in ethanol
- Dose volume: 20 uL
No. of animals per group:
twelve active dosed chambers were prepared plus two control chambers.
Control animals:
yes
Remarks:
Two control chamber were prepared
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Obtained from cosmetic surgery
- Type of skin: Female breast and abdominal skin
- Preparative technique: Freshly obtained skin, was thawed for processing. Following removal of subcutaneous fat by blunt dissection, individual portions of skin were immersed in water at 60°C for 50 secs. The skin was then pinned, dermis side down, on a cork board and the epidermis (comprising stratum corneum and epidermis) gently removed from the underlying dermis. The latter was discarded and the epidermal membrane floated onto the surface of water and was taken up onto aluminium foil. The membranes were thoroughly dried and stored flat at -20°C until used. On the day of use, the epidermal membranes were floated onto water from the aluminium foil and taken onto 25 mm diameter filter paper supports, ensuring complete coverage of the filter paper. The membranes were then mounted onto the diffusion chambers.
- Membrane integrity check: YES

PRINCIPLES OF ASSAY
- Diffusion chamber: Franz-type
- Receptor fluid: Water
- Static system: YES
- Test temperature: 32±1°C

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Total recovery:
- Total recovery: Overall recovery of radioactive material was 92±0.8%.
- Recovery of applied dose acceptable: YES
Percutaneous absorption
Dose:
20 uL/cm2 1% HHCB
Parameter:
percentage
Absorption:
ca. 5.2 %
Remarks on result:
other: 24 hrs
Remarks:
Total HHCB in remaining stratum corneum plus epidermis, filter paper, and receptor fluid

Any other information on results incl. tables

Table 1: Distribution of HHCB in all compartments at 24 hrs (% of applied dose)

  Surface wipe  Strip 1 Strips 2 -3 Strips 4 -6  Strips 7 -10  Remaining s  kin  Receptor phase  Donor chamber  Filter paper  Total recovered 
Mean 58.2  2.15  2.04  1.07  0.578  4.52  0.397  22.9  0.245  92.1 
SD  6.5  0.86  1.21  0.58  0.315  1.96  0.197  4.8  0.108  2.7 

Applicant's summary and conclusion

Conclusions:
The results of the present study indicated that the level of in-vitro percutaneous absorption of HHCB was quite low, with the majority of the applied HHCB remaining at the skin surface.
Executive summary:

The dermal absorption (non-GLP, but with QA statement) of HHCB was determined over a 24-hr period according to the methodology of the SCCNFP. Radiolabeled HHCB (uniformly labelled in the aromatic ring – radiochemical purity 99.3%) was applied in 1% solution in ethanol (96% v/v) to human epidermal membranes (prepared from female breast or abdominal skin and assayed for integrity with tritiated water) supported on a piece of filter paper (for strength) in glass diffusion cells (n=12). The area of the membrane available for absorption was approximately 1 cm2 and the average applied dose was 20±0.2 µL/cm2. The amount of material absorbed into the receptor phase, 6% Volpo N20 (to enhance solubility) in pH 7.4 phosphate buffered saline, after 24 hr was 0.40±0.06% of the applied dose. The majority of applied HHCB (81±2% of the applied dose) was found in the 24-hr surface wipe and donor chamber wash plus wipe. The stratum corneum tape strips contained 5.8±0.8% of the applied dose and the remaining stratum corneum plus epidermis 4.5±0.6% of the applied dose. Levels of HHCB in the remaining stratum corneum plus epidermis, filter paper (on which the epidermis samples rested) and permeated HHCB were combined to produce a total absorbed dose value of 5.2±0.6% of the applied dose. Overall recovery of radioactivity was 92±0.8%.