Introduction: Hair dyes may pose risks to human health due to their complex chemical composition and reactivity, potentially affecting both users and professionals, with some evidence linking permanent dyes to an increased risk of bladder cancer.
Black hair dye is a semi-permanent formulation containing multiple dyes, including azo compounds and anthraquinones. This study aimed to evaluate the cytotoxic, genotoxic and mutagenic potential of a mixture of hair dyes used in the formulation of black color (BHD), as well as its potential to alter the activities of apoptosis genes.

Methods: Human hepatoma cells (HepG2) were selected as the test organism due to their expression of phase I and II metabolizing enzymes, making them a well-established tool for evaluating mutagens and pro-mutagens. The tested concentrations were defined based on the commercial usage ratio indicated by the manufacturer (Arianor Cherry Red 0.002%, Arianor Sienna Brown 0.04% and Arianor Ebony 0.32%). In order to access DNA damage induced by BHD, a comet assay, micronucleus test and gene expression analysis were performed with HepG2 cells, while an Ames test was performed with Salmonella typhimurium

Results: Results showed cytotoxicity in HepG2 cells at concentrations allowed for human use; genotoxic effects were recorded by the comet assay for the lowest tested concentrations, and a large number of micronuclei were registered for all tested concentrations of BHD. Mutagenicity was confirmed in Salmonella typhimurium TA100 assays without S9, and altered expression was observed in two apoptosis-related genes (cytochrome c and caspase 9).

Conclusions: The findings support strategies to protect exposed populations from DNA-damaging chemicals and highlight their potential role in degenerative diseases, including cancer. Further investigation is needed on the adverse effects of hair dyes in humans and the environment.