Mechanistic-based non-animal assessment of eye toxicity: Inflammatory profile of human keratinocytes cells after exposure to eye damage/irritant agents

Eye toxicity is a mandatory parameter in human risk and safety evaluation for products including chemicals, pesticides, medicines and cosmetics. Historically, this endpoint has been evaluated using the Draize rabbit eye test, an in vivo model that was never formally validated. Due to advances in scientific knowledge, economic and ethical issues, non-animal methods based on mechanisms of toxicity are being developed and validated for increasing the capability of these models to predict eye toxicity. In this study, the Cytometric Bead Array (CBA) and ELISA assays were used to evaluate the inflammatory cytokine profile produced by HaCaT human keratinocytes after exposure to chemicals with different UN GHS eye toxicity classifications, aiming to stablish a correlation between inflammatory endpoints and eye toxicity (damage/irritation) potential. As a first step, cytotoxic profile of the chemicals, including 3 non-irritants and 10 eye toxicants (GHS Category 1, 2A and 2B), was evaluated after 24 h exposure using MTT assay and Inhibitory Concentration of 20% of cell viability (IC20) was calculated for each chemical. Then, the cells were exposed to these chemicals at IC20 for 24 h and supernatants and cell lysates were analyzed by CBA assay for quantification of the following cytokines: IL-6, IL-8, IL-10, IL-1β, TNF and IL-12p70. Regarding cytotoxicity evaluation, chemicals showed different cytotoxicity profiles and data demonstrated no correlation with their UN GHS classification. Among the cytokines evaluated, IL-1β production has changed after exposure and such alterations were confirmed by quantification employing ELISA method. The higher intracellular levels of IL-1β were found in GHS Category 1 chemicals, followed by Category 2A and 2B, while non irritants did not induce such increase. Thus, these findings show that IL-1β measurement, using HaCaT model, can be a considerable biomarker to identify chemicals according to their potential in promote eye toxicity, differentiating damage from irritation potential.