Black tattoo inks induce reactive oxygen species production correlating with aggregation of pigment nanoparticles and product brand but not with the polycyclic aromatic hydrocarbon content

Abstract

Black tattoo inks are composed of carbon nanoparticles, additives and water and may contain polycyclic aromatic hydrocarbons (PAHs). We aimed to clarify whether reactive oxygen species (ROS) induced by black inks in vitro is related to pigment chemistry, physico-chemical properties of the ink particles and the content of chemical additives and contaminants including PAHs. The study included nine brands of tattoo inks of six colours each (black, red, yellow, blue, green and white) and two additional black inks of different brands (n = 56). The ROS formation potential was determined by the dichlorofluorescein (DCFH) assay. A semiquantitative method was developed for screening extractable organic compounds in tattoo ink based on gas chromatography-mass spectrometry (GC-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Two black inks produced high amounts of ROS. Peroxyl radicals accounted for up to 72% of the free radicals generated, whereas hydroxyl radicals and H₂O₂ accounted for <14% and 16%, respectively. The same two inks aggregated strongly in water in contrast to the other black inks. They did not exhibit any shared pattern in PAHs and other organic substances. Aggregation was exclusively shared by all ink colours belonging to the same two brands. Ten of 11 black inks had PAH concentrations exceeding the European Council's recommended level, and all 11 exceeded the recommended level for benzo(a)pyrene. It is a new finding that aggregation of tattoo pigment particles correlates with ROS production and brand, independently of chemical composition including PAHs. ROS is hypothesized to be implicated in minor clinical symptoms.