The Impact of OECD Guidelines on Skin Absorption Studies
The purpose of dermal absorption studies of topical formulations is to obtain quantitative information on the amounts that can enter, under in-use conditions, into the systemic compartment. These quantities can then be taken into consideration to calculate a safety factor. Justification for the use of in vitro dermal absorption studies on isolated skin is based on the fact that the epidermis, in particular the stratum corneum, forms the principal in vivo barrier of the skin against the penetration and uptake of xenobiotics in the body.
Under in vivo conditions, the microcirculatory system (blood and lymph vessels) carries compounds from the dermis into the central compartment. In vitro, the microcirculation is eliminated. Consequently, under in vitro conditions, dermal tissue may retain penetrating compounds that, in vivo, would have been removed into the systemic compartment. Thus, either the dermis must be removed prior to in vitro investigations (epidermal membrane) or such possible in vitro retention in the dermis must be taken into account when interpreting the in vitro results. After topical application, xenobiotics detected in vitro in the skin, particularly in the stratum corneum and the pilosebaceous units, might have been lost in vivo from the skin via desquamation or sebum secretion respectively. Because these processes are not present in vitro, the final epidermal (stratum corneum) levels in vitro could be elevated compared with the corresponding in vivo levels. According to these principles, the following should be included in the protocol for in vitro dermal absorption studies:
Studies should be performed on appropriate standardized skin preparations. The respective choice should be justified in the protocol. At the end of the experiment, a full mass balance should be performed.
When considerable cutaneous metabolism of the ingredients to be tested occurs in vivo, further studies are necessary. It should be noticed that frozen skin preparations lack the enzyme systems to biotransform the test compound and do not provide an appropriate picture of the potential metabolites and their dermal absorption. Therefore, in vitro studies using frozen skin do not provide information on the dermal absorption of a substance; neither do they give information on the metabolite(s) of the test material that undergoes extensive biotransformation.
Whenever irreversible binding of an ingredient to the epidermis followed by elimination through in vivo desquamation of the skin surface is assumed, this must be documented by separate experiments.
At present, two OECD Guidelines on dermal absorption are available, namely OECD Draft Guideline 427 on skin absorption: in vivo method [OECD 427], and OECD Draft Guideline 428 on skin absorption: in vitro method [OECD 428].
Over the years, practical experience has been gained in using the in vitro methodology: in vitro dermal absorption studies are now currently used and results are incorporated in toxicological dossiers of cosmetic ingredients, plant protection products and biocides,
At present, it is only possible to use skin preparations of human cadaver skin (or excised human skin from plastic surgery). The quality of cultured or reconstituted skin has improved importantly during the last year, but does not yet represent the full barrier function as known in vivo. The OECD Guideline 428 should be followed as close as possible, taking into account the guidance given here. Any deviation from these should be accompanied by a specific justification of the particular changes made and appropriate scientific references should be mentioned.
The test substance is applied in an appropriate formulation on the skin sample which usually is placed in a diffusion cell. The skin is positioned between the upper and lower chambers of the cell. The latter may be either of static or flow-through design. The integrity of the barrier should be verified by an appropriate method. The test sample remains in contact with the skin on the donor side for a defined period of time (leave-on or rinse-off respectively, depending on the intended use conditions). The receptor fluid may be sampled once at the end of the experiment or preferably at various time points before the end so that an absorption profile may be constructed. A justification of the procedure used (static or flow-through conditions) should be provided. The skin and/or fluid samples are analyzed by appropriate and validated analytical methods (scintillation counting, HPLC/UPLC, LC-MS, or GC) of which the sensitivity for the particular ingredient under investigation, should be mentioned.
Skin absorption is affected by several factors: for example: physical and chemical properties of the substance, type and composition of the formulation, occlusion, concentration within the formulation, exposure pattern, skin site of the body
OECD Guideline 428 provides guidance on the factors that may affect dermal absorption using a common in vitro methodology and describes tests and procedures for such studies:
- Diffusion cell
- Receptor fluid
- Skin preparation
- Skin integrity
- Application to the skin
- Duration of exposure and sampling
- Analytical methods
- Data analysis and test report
Barrier integrity is crucial for the experiment, and must therefore be investigated. This is achieved by either measuring the penetration of a marker molecule, e.g. tritiated water, caffeine or sucrose, or by physical methods like TEWL (Transepidermal Water Loss) or TER (Transcutaneous Electrical Resistance) measurements. Data obtained should be reported.
Appropriate analytical techniques, e.g. scintillation counting, HPLC/UPLC, LC-MS/MS or GC, should be used. Their validity, sensitivity and detection limits should be documented in the report.
The mass balance of the applied dose must also be determined with the data mentioned above.
The overall recovery of test substance (including its metabolites) should be within the range of 85-115%. If lower recoveries of the test substance are obtained, the reasons need to be investigated and explained.
The variability of dermal absorption studies depends on the penetration rate of a particular ingredient; the lower the penetration rate, the higher the variability. This high variability is due to known intra-individual and inter-individual characteristics of the stratum corneum barrier. The relative variability of the method should be documented. The validity of the method used should be assessed at regular intervals by including penetrating compounds like caffeine or benzoic acid as reference substances. These data should be included in the study report. The reproducibility of the method should be shown by the use of minimum six evaluable samples (human or pig skin), from each of at least 3 donors per dose. The coefficient of variation should be less than 30%. If statistical evaluation is not possible, the highest observed penetration value will be used in the systemic exposure dosage (SED) calculation.
The doses as well as the contact time (exposure) with the skin are chosen to mimic intended use conditions. The amount of the formulation to be applied is adapted to the consumer use values described in the notes of the Guidance and usually lies between 2-5 mg/cm2 for solids and semi-solid preparations, and up to 10 μl/cm2 for liquids. Deviations should be explained. The volume of formulation used should be appropriate to spread the sample homogeneously on the skin surface. This depends strongly on the viscosity of the formulation.
Amounts of the test compound must be determined in:
the surplus on the skin
the stratum corneum (e.g. adhesive tape strips)
the epidermis without stratum corneum
the receptor fluid
It is necessary to check for substance adsorbed in the equipment. The amounts of penetrated substance(s) found in the receptor fluid are considered to be systemically available. The epidermis (except for the stratum corneum) and dermis are considered as a sink; therefore, the amounts found in these tissues are equally considered as absorbed and are added to those found in the receptor fluid. The amounts that are retained by the stratum corneum at the time of sampling are not considered to be dermally absorbed, and thus they do not contribute to the systemic dose.