Adaption of in vitro and in chemico phototoxicity tests for tattoo pigments and the effect of adsorption of the phototoxic contaminant benzo[a]pyrene to carbon black

Phototoxicity plays an important role in tattoo toxicology due to the skin's daily exposure to sunlight and the permanent deposition of pigments in skin. Here, we investigated in chemico reactive oxygen species generation using 2′,7′-dichlorodihydrofluorescein diacetate and in vitro phototoxicity towards skin fibroblasts based on the OECD Test Guideline 432.

Proficiency substances, titanium dioxide (TiO₂, anatase 30 nm, anatase 100 nm, rutile 30 nm), pigment red 22 (PR 22) with and without purification and carbon black (CB) were investigated. Phototoxic properties of benzo[a]pyrene (B[a]P) were investigated with and without adsorption to CB. Pigments were characterised by dynamic light scattering.

All proficiency substances except for norfloxacin were predicted successfully and only TiO₂ anatase exhibited phototoxicity in the cell-based approach. B[a]P showed the expected phototoxicity in contrast to B[a]P bound to carbon black. Upon UVA irradiation, only TiO₂ pigment anatase and PR 22 generated ROS in the cell-free assay. DLS measurements showed a high degree of polydispersity/agglomeration.

OECD Test Guideline 432 should be applied with an incubation time of 24 h when testing highly insoluble pigments. The in chemico ROS assay introduced below might serve as a quick screening test but may be further adapted to predict the in vitro outcome.