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Test method for evaluating the photocytotoxic potential of fluorescence imaging products.

Various fluorescence imaging agents are currently under clinical studies. Despite significant benefits, phototoxicity is a barrier to the clinical translation of fluorophores. Current regulatory guidelines on medication-based phototoxicity focus on skin effects during sun exposure. However, with systemic and local administration of fluorophores and targeted illumination, there is now possibility of photochemical damage to deeper tissues during intraoperative imaging procedures. Hence, independent knowledge regarding phototoxicity is required to facilitate the development of fluorescence imaging products. Previously, we studied a cell-free assay for initial screening of reactive molecular species generation from fluorophores. The current work addresses a safety test method based on cell viability as an adjunct and a comparator with the cell-free assay. Our goal is to modify and implement an approach based on the in vitro 3T3 neutral red uptake assay of the Organization for Economic Co-Operation and Development Test Guideline 432 (OECD TG432) to evaluate the photocytotoxicity of clinically relevant fluorophores. These included indocyanine green (ICG), proflavine, methylene blue (MB), and IRDye800, as well as control photosensitizers, benzoporphyrin derivative (BPD) and rose bengal (RB). We performed measurements at agent concentrations and illumination parameters used for clinic imaging. Our results aligned with prior studies, indicating photocytotoxicity in RB and BPD and an absence of reactivity for ICG and IRDye800. DNA interactive agents, proflavine and MB, exhibited drug/light dose-response curves like photosensitizers. This study provides evidence and insights into practices useful for testing the photochemical safety of fluorescence imaging products.

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