Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
In vitro predictions of skin absorption of caffeine, testosterone, and benzoic acid: a multi-centre comparison study
Author:
van de Sandt
Year:
2004
Bibliographic source:
Regulatory Toxicology and Pharamacology 39, 271-281

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
GLP compliance:
yes
Remarks:
Two of ten participants were GLPcompliant while the other laboratories adhered to this quality system as much as possible.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): caffeine, [1-methyl-14C]caffeine
Radiolabelling:
yes
Remarks:
[1-methyl-14C]caffeine (51.2mCi/ mmol)

Test animals

Species:
other: rat and human skin
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 weeks

ENVIRONMENTAL CONDITIONS
- no data

Administration / exposure

Type of coverage:
not specified
Vehicle:
other: ethanol/water (1:1, v/v)
Duration of exposure:
24 h
Doses:
- Nominal doses: 4.0 mg/mL ethanol/water
- Dose volume: The application volume was 25 µL/cm² which is considered the minimum volume necessary to produce a homogeneous distribution on the skin surface. This represented a finite dose (100 µg/cm²), in order to mimic occupationally relevant situations.
- Rationale for dose selection: All participating laboratories performed their studies according to a detailed study protocol in which the experimental
design and parameters such as the dose of the test chemical.
No. of animals per group:
No data
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions:
The dose solutions were prepared freshly by each laboratory in ethanol/water (1:1, v/v), yielding a concentration of 4.0 mg/mL for each compound.
Participants with a license to handle radiochemicals prepared the dose solutions by mixing appropriate amounts of radiolabelled and non-radiolabelled test substances. The dose solutions were measured for exact total radioactivity prior to and directly after the application to the skin membranes.
The radioactive concentration was approximately 1MBq/mL for testosterone and caffeine
- Method of storage: No data

APPLICATION OF DOSE: 100 µg/cm²

ANALYSIS
Method type(s) for identification
- Liquid scintillation counting:
Radioactivity measurements were made by individual participating laboratories. Radioactivity in the various samples (receptor fluid, skin, skin swabs, and cell washings) was determined by liquid scintillation counting. Receptor fluid samples were added directly to an appropriate scintillation fluid. For analysis of the skin membranes, an aliquot of the tissue digest (1.5M KOH in 20% aqueous ethanol) was used.
- Caculations:
The percentage of the dose recovered in the receptor fluid in 24 h, the percentage in the skin membrane, and the percentage total recovery (for radiolabelled studies) was calculated.
The presence of the test compound in the skin membrane after washing the application area at 24 h was expressed by the ratio between
the percentage of the dose in skin and receptor fluid [total penetration (TP)] and the percentage of the dose in receptor fluid (RF).

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: 9 different european laboratories, as specified in the publication.
- Ethical approval if human skin: The supply and use of human and animal tissue was in full accordance with national ethical guidelines
- Type of skin: breast, leg, abdomen,
- Preparative technique: Both human and rat skin membranes were prepared from frozen skin. Whole skin was cleaned of subcutaneous
fat and the skin was stored.
- Thickness of skin (in mm): Most laboratories used human skin with a thickness between 0.7 - 1.1 mm, while one participant used skin that was 0.8 -
1.8 mm. Three laboratories used dermatomed skin with a thickness of 0.5 - 0.7mm or 0.3 - 0.4mm (1 participant). The range of skin thickness used by the various laboratories allowed for the assessment of the influence of skin thickness on the absorption characteristics of the test compounds.
Skin from more than one donor was used in each experiment and each experimental group consisted of 5 -7 skin membranes form different individuals.
Rat full-thickness skin was used by one participant and was collected from the back (clipped carefully) of four weeks old male Sprague Dawley rats.
- Membrane integrity check: After skin membranes were thawed and mounted in the diffusion cell, the skin integrity was assessed by either visual assessment, permeation of tritiated water (cut-off Kp > 3.5 x 10e-3 cm/h) or capacitance (cut-off: 55 nF), depending on the laboratory.
- Storage conditions: approximately -20°C (two participants at approximately -70°C) for a maximum period of one year.
- Justification of species, anatomical site and preparative technique: For comparison of the results, all laboratories performed their studies
according to a detailed study protocol in which the experimental design and parameters such as the dose of the test chemical, vehicle, duration of the experiment, preparation of the skin membranes, receptor fluid type, occlusion, temperature, sampling times, and number of replicates were defined.

PRINCIPLES OF ASSAY
- Diffusion cell: different diffusion cell systems were used: flow-through (6 laboratories), static (4 laboratories)
- Receptor fluid: receptor fluid consisted of saline (0.9% NaCl), 0.2 - 3.5 mL, with or without stirrer.
Aliquots of the receptor fluid were collected at various time points (minimally at 1, 2, 4, 8, and 24 h post-dosing).
- Solubility of test substance in receptor fluid: No data
- Static system: For static cells, the original volume of the receptor fluid was restored by adding fresh receptor fluid to the receptor compartment directly after each sampling. In case of non-radiolabelled test compounds, the receptor fluid samples were stored at approximately -20°C until analysis.
- Flow-through system: For systems using flow-through diffusion cells, the flow of receptor fluid was approximately 1.5 mL/h.
- Test temperature: No data
- Humidity: No data
- Occlusion: During exposure the donor compartment remained occluded.
- Reference substance(s): testosterone, caffeine, and benzoic acid

Results and discussion

Absorption in different matrices:
- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
The mean maximum absorption rate of caffeine through human skin membranes was 2.24 ± 1.43 µg/cm²/h, while the amount in the receptor fluid after 24 h was 24.5 ± 11.6% of the dose applied (9 laboratories). The mean maximum absorption rate of caffeine through rat skin (1 laboratory) was 6.82 µg/cm²/h and the amount in the receptor fluid after 24 h was 53.7%.
- Skin preparation (in vitro test system):
For both human and rat skin, the ratio TP:RF was only slightly higher than 1.0, indicating that only a small amount caffeine remained in the skin membrane after washing the application area.
The CV value of the maximum absorption rate varied from 12.0% to 91.4%. For the percentage in the receptor fluid (at 24 h), the CV values
ranged between 5.4% and 66.0%.
- Stratum corneum (in vitro test system): (i.e tape strips)
Total recovery:
- Total recovery: The total recovery of the radioactivity ranged between 66.4 and 100.6% (7 laboratories).
- Recovery of applied dose acceptable:
- Results adjusted for incomplete recovery of the applied dose: No data
- Limit of detection (LOD): No data
- Quantification of values below LOD or LOQ: No data
Percutaneous absorption
Dose:
4 mg/L
Parameter:
percentage
Absorption:
ca. 25.7 %
Remarks on result:
other: 24 h
Conversion factor human vs. animal skin:
In rat total penetration was 61.3 % of the dose

Applicant's summary and conclusion