In vitro benzyl alcohol cytotoxicity: Implications for intravitreal use of triamcinolone acetonide

Abstract

The aim of the study was to investigate the toxicity of benzyl alcohol (BA), the preservative in commercial triamcinolone acetonide (TA) suspensions, on retinal pigment epithelial (RPE) cells. Cultured RPE cells from a human cell line (ARPE-19) and from rabbits were exposed to the balanced salt solution (control) or BA (0.0225, 0.225, 0.9, 3 or 9 mg/mL) for 5, 30, 60, or 120 min. Morphological changes of RPE cells were evaluated by the trypan blue in situ staining. The proportions of dead cells were quantitatively measured by the trypan blue exclusion assay, and those of functional cells were assessed by a mitochondrial dehydrogenase assay. The mechanism of cytotoxicity was determined by the acridine orange/ethidium bromide staining and DNA laddering technique. Furthermore, ultrastructural changes were observed by transmission electron microscopy. The results showed that RPE cell damage was dose- and time-dependent. BA 0.225 mg/mL, the clinically relevant concentration in TA following intravitreal injection, caused ultrastructural damage and impaired human RPE cell function at 2 h; but BA 0.0225 mg/mL did not. BA 9.0 mg/mL, the concentration in commercial TA suspensions, was toxic within 5 min on each assay for both human and rabbit RPE cells. The major mechanism of cell death was necrosis. In conclusion, BA in commercial TA suspensions injected intravitreally (0.225–9 mg/mL) can damage RPE cells. Our in vitro study on benzyl alcohol cytotoxicity has significant clinical implications for intravitreal use of TA. We suggest that, before a commercial TA solution is used intravitreally, the vehicle should be removed to prevent damaging the RPE layer, particularly during macular hole surgery. Commercial development of a preservative-free TA suspension for intraocular use is urged.