Percutaneous Penetration and Metabolism of 2-Nitro-p-phenylenediamine in Human and Fuzzy Rat Skin

Abstract

2-Nitro-p-phenylenediamine (2NPPD) is a dye used in semipermanent and permanent (tinting color) hair dye formulations. National Toxicology Program toxicology and carcinogenesis testing of 2NPPD has raised concerns about its safety. Therefore, we initiated in vitro studies to measure absorption and metabolism of 2NPPD in human and fuzzy rat skin and rat jejunal tissue. Intestinal tissue metabolism of 2NPPD was compared to skin metabolism since toxicology data from oral 2NPPD studies will be used for future safety assessment purposes. Absorption was measured over 24 h by using flow-through diffusion cells with a receptor fluid consisting of Hepes-buffered Hank's balanced salt solution. Dosing vehicles were applied to skin and intestine in the diffusion cells for 30 min. 2NPPD metabolites were determined by high-performance liquid chromatography methodology. In human skin, the percentages of total applied dose absorbed (receptor fluid + skin) over 24 h were 9.2 ± 5.7 (mean ± SD) and 9.5 ± 3.2 for the ethanol and semipermanent vehicles, respectively, with approximately 3% remaining in skin. In rat skin, the percentages of total applied dose absorbed over 24 h were 9.3 ± 1.2 (mean ± SE), 6.9 ± 1.2, and 4.2 ± 0.1 for the ethanol, semipermanent, and permanent formulation vehicles, respectively, with approximately 3% remaining in skin. In rat intestinal tissue, the percentage of total applied dose absorbed over 24 h was 10.9 ± 1.2, with approximately 5% remaining in the tissue. In human and rat skin, 2NPPD was metabolized to triaminobenzene and N4-acetyl-2NPPD. 2NPPD was also metabolized to a sulfated 2NPPD metabolite in rat skin, but not in human skin. 2NPPD was extensively metabolized in both human and rat skin with ethanol application; metabolism was not as extensive with a semipermanent formulation application. In rat intestinal tissue, 62% of 2NPPD was metabolized upon absorption to triaminobenzene and N4-acetyl-2NPPD. Differences in the metabolic profiles (proportion of each metabolite formed) were found between the skin and intestinal tissue. These results suggest that 2NPPD is rapidly absorbed and extensively metabolized in both skin and intestinal tissue. The extent of metabolism and the metabolic profile were found to be species-, tissue-, and dosing vehicle-dependent. Metabolism information will be useful in predicting the extent of 2NPPD and/or 2NPPD metabolite systemic absorption relative to a dermal exposure, which will improve the health hazard assessment of 2NPPD.