Assessment of Excised and Reconstructed Human Pigmented Skin Models for In Vitro Permeation Testing

Percutaneous absorption is essential in the safety assessment of topical drugs and cosmetic products, but the potential impact of skin pigmentation on dermal absorption remains understudied. Here, a novel framework integrating quantitative pigmentation characterization, barrier function assessment, and in vitro permeation testing (IVPT) to investigate the role of melanin in percutaneous absorption using both excised and reconstructed human skin models was established. Abdominal excised human skin (EHS) from female donors of Fitzpatrick phototypes (FP) I/II and FP V/VI and reconstructed human pigmented epidermis (RHPE) with melanocytes derived from Caucasian and African American donors were tested. RHPEs were cultured for 15 days in basal or melanogenesis-stimulating media. Skin pigmentation level, transepidermal water loss (TEWL), and the in vitro permeation of radiolabeled caffeine, salicylic acid, testosterone, mannitol, hydroquinone, and ciprofloxacin were evaluated in EHS and RHPE samples. Melanin quantification assays and histological analyses confirmed higher pigmentation in EHS of FP V/VI versus FP I/II donors, in RHPE from African American versus Caucasian donors, and in RHPEs cultured under melanogenesis-stimulating versus basal conditions, especially in tissues from African American donors. No significant differences in TEWL were observed between EHS groups or across RHPE groups. In general, no statistically significant differences were observed in the cumulative amount, flux, and mass distribution of test chemicals between EHS groups or across RHPE groups. The study demonstrated the feasibility of using a standardized toolkit and pigmented human skin models to investigate the role of melanin in percutaneous absorption in vitro. The ability to modulate pigmentation levels in RHPEs within the same donor background supports their utility as a reproducible model for evaluating melanin-associated variability in dermal safety assessments, particularly for compounds that may interact with melanin.