Comparative neurotoxic effects of conventional and alternative plasticizers on microglia

Plasticizers are widely incorporated into plastics to enhance their flexibility, yet their non-covalent binding to the polymer matrix enables continuous human exposure through multiple routes. Increasing evidence suggests that some of these compounds can reach the central nervous system. One of the most commercially produced, di(2-ethylhexyl) phthalate (DEHP), is currently restricted due to documented toxicological effects, including endocrine disruption and neurotoxicity. This has driven its progressive substitution with alternative compounds considered safer such as acetyl tributyl citrate (ATBC) and acetyl triethyl citrate (ATEC), although accumulating evidence suggests that these substitutes may induce similar neurobiological effects. More recently, bio-based plasticizers such as thymol:menthol (TY-ME), a natural deep eutectic solvent (NADES), have been proposed as next-generation alternatives. These systems are considered biodegradable and potentially biocompatible and may even exert intrinsic bioactive properties; however, their impact on neural cells remains largely unexplored.

In this study, we evaluated the effects of these plasticizers on human microglial HMC3 cells, focusing on cell viability and molecular responses associated with neuroinflammation and oxidative stress. Cytotoxicity was assessed using the MTT assay, while gene expression levels of TGF-β1, IL-6, IL-1β, and HMOX1 were quantified by RT-qPCR. DEHP exposure resulted in pronounced cytotoxicity together with strong induction of pro-inflammatory and oxidative stress markers. ATBC elicited a comparable response profile, suggesting that its biological impact may resemble that of the phthalate it replaces. In contrast, TY-ME exhibited moderate cytotoxic effects and minimal modulation of gene expression, indicating a comparatively attenuated impact on microglial activation. ATEC showed the lowest overall toxicity, although it was associated with a detectable oxidative stress response.

Overall, these findings highlight that phthalate-free alternatives are not necessarily devoid of biological activity and underscore the importance of systematically evaluating both conventional and emerging plasticizers in relevant human cell models to support the development of safer materials.