An OECD TG 428 study ring trial with 14C-Caffeine demonstrating repeatability and robustness of the dermal absorption in vitro method.

The dermal absorption potential of 14C-Caffeine applied as a 4 mg/mL concentration (10 µL/cm2 finite dose) was investigated in six laboratories under Good Laboratory Practice conditions using an OECD TG 428-compliant in vitro assay with flow-through cells and split-thickness human skin. Potential sources of variation were reduced by a standardized protocol, test item and skin source. Particularly, skin samples from same donors were distributed over two repeats and between labs in a non-random, stratified design. Very similar recovery was achieved in the various assay compartments between laboratories, repeats and donors, demonstrating that the assay can be robustly and reliably performed. The absorption in one laboratory was 5-fold higher than in the others. This did not clearly correlate with skin integrity parameters but might be associated with an accidental COVID-19 pandemic-related interruption in sample shipment. It is possible that other factors may affect dermal absorption variation not routinely assessed or considered in the current method. The mean receptor fluid recovery, potential absorption (recovery in receptor fluid and skin except tape strips 1 and 2) and mass balance of caffeine was 6.99%, 7.14% and 99.13%, respectively, across all and 3.87%, 3.96% and 99.00% in the subset of five laboratories.

Application of in vitro skin penetration measurements to confirm and refine the quantitative skin sensitization risk assessment of methylisothiazolinone.

Use of quantitative risk assessment (QRA) for assessing the skin sensitization potential of chemicals present in consumer products requires an understanding of hazard and product exposure. In the absence of data, consumer exposure is based on relevant habits and practices and assumes 100% skin uptake of the applied dose. To confirm and refine the exposure, a novel design for in vitro skin exposure measurements was conducted with the preservative, methylisothiazolinone (MI), in beauty care (BC) and household care (HHC) products using realistic consumer exposure conditions. A difference between measured exposure levels (MELs) for MI in leave-on versus rinse-off BC products, and lower MELs for MI in HHC rinse-off compared to BC products was demonstrated. For repeated product applications, the measured exposure was lower than estimations based on summation of applied amounts. Compared to rinse-off products, leave-on applications resulted in higher MELs, correlating with the higher incidences of allergic contact dermatitis associated with those product types. Lower MELs for MI in rinse-off products indicate a lower likelihood to induce skin sensitization, also after multiple daily applications. These in vitro skin exposure measurements indicate conservatism of default exposure estimates applied in skin sensitization QRA and might be helpful in future risk assessments.

An in vitro skin penetration model for compromised skin: estimating penetration of polyethylene glycol [¹4C]-PEG-7 phosphate.

BACKGROUND/AIMS: Establishing dermal penetration rates is important to better understand the safety of topically applied materials, especially for premature infant skin with compromised skin barrier function. Skin prematurity involves thinner stratum corneum and underdeveloped epidermis/dermis resulting in decreased barrier function, higher transepidermal water loss and greater chemical penetration, when compared to healthy full-term neonate/adult skin. METHODS: We developed an in vitro skin penetration model using human ex vivo skin to estimate penetration for premature/compromised skin barrier conditions by tape stripping. Skin barrier deficiency was characterized by transepidermal water loss. Baby wipe lotion containing 5 mg/cm(2) [(14)C]-PEG-7 phosphate was applied 5 times to human skin samples of intact, moderately or highly compromised skin barrier and once at 25 mg/cm(2) over 24 h. RESULTS: Overall penetration of [(14)C]-PEG-7 phosphate was low (<5%) even for highly compromised skin. The absorption rate was higher (p < 0.001) for compromised skin versus intact skin. No significant difference was seen between moderately and highly compromised skin by repeated dosing. Under single-dose conditions, penetration through highly compromised skin was significantly higher compared to intact skin (p = 0.001). CONCLUSION: Our model demonstrates that even under highly compromised skin conditions, penetration of [(14)C]-PEG-7 phosphate is low (<5%) and only 4-6 times higher compared to mature/intact skin and does not approach 100%. Penetration was unaffected by single or multiple dosing conditions.

Visualisation of drug distribution in skin using correlative optical spectroscopy and mass spectrometry imaging.

A correlative methodology for label-free chemical imaging of soft tissue has been developed, combining non-linear optical spectroscopies and mass spectrometry to achieve sub-micron spatial resolution and critically improved drug detection sensitivity. The approach was applied to visualise the kinetics of drug reservoir formation within human skin following in vitro topical treatment with a commercial diclofenac gel. Non-destructive optical spectroscopic techniques, namely stimulated Raman scattering, second harmonic generation and two photon fluorescence microscopies, were used to provide chemical and structural contrast. The same tissue sections were subsequently analysed by secondary ion mass spectrometry, which offered higher sensitivity for diclofenac detection throughout the epidermis and dermis. A method was developed to combine the optical and mass spectrometric datasets using image registration techniques. The label-free, high-resolution visualisation of tissue structure coupled with sensitive chemical detection offers a powerful method for drug biodistribution studies in the skin that impact directly on topical pharmaceutical product development.

Optimization of topical formulations using a combination of in vitro methods to quantify the transdermal passive diffusion of drugs.

This paper describes a new approach to the early-stage optimization of topical products and selection of lead formulation candidates. It demonstrates the application of open flow microperfusion in vitro in conjunction with the Franz diffusion cell to compare time-resolved, 24-hour profiles of diclofenac passive diffusion through all skin layers (including the skin barrier, dermis, and subcutis) resulting from nine topical formulations of different composition. The technique was successfully validated for in vitro sampling of diclofenac in interstitial fluid. A multi-compartmental model integrating the two datasets was analyzed and revealed that the passive diffusion of diclofenac through the dermis and subcutis does not correlate with its diffusion through the skin barrier and cannot be predicted using Franz diffusion cell data alone. The combined application of the two techniques provides a new, convenient tool for product development and selection enabling the comparison of topical formulation candidates and their impact on drug delivery through all skin layers. This approach can also generate the experimental data required to improve the robustness of mechanistic PBPK models, and when combined with clinical sampling via open flow microperfusion - for the development of better in vivo-in vitro correlative models.

Visualization of Epidermal Reservoir Formation from Topical Diclofenac Gels by Raman Spectroscopy.

PURPOSE: This work investigated whether topical pain relief diclofenac gels can form a diclofenac reservoir in the epidermal and dermal layers of human skin. METHODS: Excised human skin samples were treated with three topical diclofenac gels ex vivo and examined using Raman microscopy of transversally microtomed sections. The relative diclofenac concentration in the skin layers was calculated as the ratio of the integrated areas of bands characteristic of diclofenac (~445 cm-1) and skin (Amide I). A customized masking algorithm ensured that only diclofenac-specific signal was mapped in the resulting Raman images. RESULTS: A heterogenous spatial distribution of diclofenac was clearly visible in both the epidermis and the dermis in all samples, with a markedly higher diclofenac relative content and number of pixels above the detection limit in the epidermis compared to the dermis. CONCLUSION: The Raman images evidenced that the studied topical gels deliver diclofenac through the stratum corneum skin barrier and form a drug depot localized in the epidermis. The data are in line with earlier clinical findings that this depot acts like a true reservoir and enables sustained drug release.

Determining epidermal disposition kinetics for use in an integrated nonanimal approach to skin sensitization risk assessment.

Development of risk assessment methods for skin sensitization in the absence of toxicological data generated in animals represents a major scientific and technical challenge. The first step in human skin sensitization induction is the transport of sensitizer from the applied dose on the skin surface to the epidermis, where innate immune activation occurs. Building on the previous development of a time course in vitro human skin permeation assay, new kinetic data for 10 sensitizers and 2 nonsensitizers are reported. Multicompartmental modeling has been applied to analyze the data and determine candidate dose parameters for use in integrated risk assessment methods: the area under the curve (AUC) and maximum concentration (C(max)) in the epidermis. A model with two skin compartments, representing the stratum corneum and viable skin (epidermis and dermis), was chosen following a formal model selection process. Estimates of the uncertainty, as well as average values of the epidermal disposition kinetics parameters, were made by fitting to the time course skin permeation data from individual skin donors. A potential reduced time course method is proposed based on two time points at 4 and 24 h, which gives results close to those from the full time course for the current data sets. The time course data presented in this work have been provided as a resource for development of predictive in silico skin permeation models.