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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Percutaneous Absorption of Sunscreens in vitro: Interspecies Comparison, Skin Models and Reproducibility Aspects
Author:
Benech-Kieffer F., Wegrich P., Schwarzenbach R., Klecak G., Weber T., Leclaire J. and Schaefer H.
Year:
2000
Bibliographic source:
Skin Pharmacology and Applied Skin Physiology; 13 :324-335

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Radiolabelling:
yes

Test animals

Species:
other: pig and human
Strain:
not specified
Sex:
not specified

Administration / exposure

Type of coverage:
open
Vehicle:
other: oil-in-water emulsion
Duration of exposure:
16 h
Doses:
The test substance was incorporated in a typical substantive oil-in-water (O/W) emulsion at 5 % (w/w). 2 mg/cm² of the test substance was applied to the surface of the skin samples.
No. of animals per group:
- human skin: three skin donors (one replicate per donor) were used per test compound
- pig skin: experiments were performed on the skin from the same animal. Two to four replicates were performed per test compound.
Control animals:
no
Details on study design:
PRINCIPLES OF ASSAY
- Diffusion cell: skin samples were mounted in static diffusion cells with a surface of 2 cm² and a receptor volume of 3 mL.
- Receptor fluid: The receptor fluid was a Dulbecco modified phosphate-buffered saline solution containing 4 % (w/w) polyethylene glycol 20 oleyl ether.
- Static system: The static system was chosen because of the low absorptive properties of the sunscreens. The skin samples were placed as a barrier between the two halves of the diffusion cell; the dermal side of the skin was exposed to the receptor fluid and the stratum corneum was exposed to the air.
- Test temperature: The surface of the skin was kept at 32 ± 1 °C by thermostating the receptor solution.
- Occlusion: nonocclusive conditions
- Reference substance: salicylic acid (SA)
- Samples:
Full thickness samples: amount of tested compounds was estimated in (1) skin surface, (2) stratum corneum, (3) viable epidermis, (4) dermis and (5) receptor fluid .
Stratum corneum was separated from the rest of the epidermis by tape-stripping. Fifteen to twenty strips of the compound-treated site were made applying a constant pressure on the skin surface
(150 g/cm2). The complete removal of the stratum corneum was checked visually with the aid of a stereomicroscope.
Epidermis was isolated from the dermis by heat by holding a hair dryer over the epidermal surface . Then, the epidermis was gently peeled off the dermis with a surgical blade.
Split thickness samples: amount of tested compounds was estimated in (1) skin surface, (2) epidermis, (3) dermis and (4) receptor fluid.
Total epidermis (including the stratum corneum) was separated from the dermis
- Analysis: Liquid scintillation counting
Details on in vitro test system (if applicable):
SKIN PREPARATION
Source of skin:
- Abdominal human skin (HS) was obtained from surgical patients (Clinique du Sud, Thiais, France).
- The used pig skin (PS) was obtained from the flank of a domestic female pig from a slaughterhouse (Basel, Switzerland).

- Type of skin:
Preparations of skin membranes:
- Human skin: In the laboratory, all skin samples were rinsed with de-ionized water and checked visually to ensure that they were unaltered by preparation for surgery or by surgical excision. The subcutaneous fat was removed, then the skin samples were put in plastic bags, sealed and stored at -20 °C until the day of experiment (up to 6 months). Three skin donors (one replicate per donor) were used per tested compound.
- Pig skin: The skin was cleaned and shaven and the skin samples were sealed and stored at -20 °C until use. The experiments in both laboratories were performed on skin from the same animal. Two to four replicates were performed per tested compound.
- Skin samples were thawed at room temperature and prepared on the day of the experiment.

Thickness of skin:
- Full-thickness PS: 2,500-3,500 µm (directly mounted in diffusion cells)
- Full-thickness HS: 1,400-2,200 μm (directly mounted in diffusion cells)
- Split-thickness: skin membranes were prepared with an electric dermatome (Davies Simplex, Thackray Surgery, UK). Skin samples were fixed on a dissection board. Epidermal side up, and sections were cut at 300-500 µm. The dermatomed layer thus obtained included the epidermis and some dermal tissue.

Membrane integrity check:
- Skin integrity was checked visually with the aid of a stereomicroscope (Wild M8, Leica, Switzerland).

Justification of species and preparative technique:
- Data from the literature suggest that the domestic pig is the best animal model to use, as pig skin (PS) has been demonstrated to have histological, physiological and permeability properties similar to those of HS.
- full-thickness PS was compared with split-thickness PS (including the whole epidermis and the upper dermis) to investigate the possibility that the presence of a dermal membrane represents a significant additional 'artificial' barrier in in vitro studies, especially for lipophilic molecules.

Results and discussion

Percutaneous absorptionopen allclose all
Dose:
2 mg/cm² (test substance)
Parameter:
percentage
Absorption:
4.03 %
Remarks on result:
other: 16 h
Remarks:
pig skin (split thickness) mean total skin, n=3, SE +/- 2.21
Dose:
2 mg/cm² (test substance)
Parameter:
percentage
Absorption:
2.63 %
Remarks on result:
other: 16 h
Remarks:
pig skin (full thickness): mean total skin, n=3 , SE +/- 1.05
Dose:
2 mg/cm² (test substance)
Parameter:
percentage
Absorption:
6.18 %
Remarks on result:
other: 16 h
Remarks:
human skin (split thickness): mean total skin, n=3 , SE +/- 3.90

Any other information on results incl. tables

Split thickness skin pig human
  % applied dose  +/- SE % applied dose  +/- SE
Skin surface 92.6 1.18 94.0 4.24
Stratum corneum + Epidermis  2.11 1.53 5.12 3.20
Dermis 1.71 0.75 0.99 0.67
Recepor fluid 0.21 0.09 0.06 0.06
Total skin*  4.03 2.21 6.18 3.90
Recovery 96.7 1.25 100 7.22

*SC+Epidermis+Dermis+Receptor fluid

Full thickness skin pig
  % applied dose  +/- SE
Skin surface 92.1 8.43
Stratum corneum + Epidermis  2.41 0.91
Dermis 0.22 0.13
Recepor fluid 0.004 0.002
Total skin*  2.63 1.05
Recovery 94.7 9.44

*SC+Epidermis+Dermis+Receptor fluid

Table: analytical control of radiolabelled O/W emulsion (means +/- SE, n=3)

 

Test substance

Total concentration, % (w/w)

4.64 +/- 0.01

Radioactive concentration, MBq/g

34.15 +/- 0.18

Applicant's summary and conclusion