H-Content Is Not Predictive of Perfluorocarbon Ocular Endotamponade Cytotoxicity in Vitro

Abstract

In recent years, cases of retinal toxicity occurred in some European, Middle Eastern, and South American countries following the use of perfluorocarbon liquids (PFCLs) on vitreoretinal surgeries owing to impurities in the product. Moreover, Spanish ophthalmologists reported several toxic cases on the use of perfluoro-n-octane Ala Octa (Alamedics, Dornstadt, Germany), raising the necessity of reviewing the current validated methods used for assessing the safety of PFCLs. We proved that in samples of PFCLs contaminated on purpose with impurities previously detected in Ala Octa devices, the determination of the so-called H-content using a ¹H NMR quantitative assay implemented with the electronic reference to access in vivo concentrations 2 technology failed to demonstrate a correlation between the H-content and in vitro cytotoxicity test in ARPE-19 and BALB 3T3 cell lines. Therefore, direct information on the safety of PFCLs was provided only by the cytotoxicity test in vitro validated according to ISO 10993-5, and the H-content was not predictive of perfluorocarbon ocular endotamponade cytotoxicity in vitro.

Figure 1

Correlation between H-content of analyzed PFO and PFD samples with cytotoxicity determined in ARPE-19 (left panel) and BALB-3T3 (right panel) cells. Shaded areas show the confidence intervals for the fitted lines. A significant correlation was found for both cell lines (for ARPE-19 cells: r = 0.8820, p < 0.0001; for BALB-3T3 cells: r = 0.9718, p < 0.0001).

Figure 2

Dendrograms showing hierarchical clustering (Ward’s method) of samples according to cytotoxicity and H-content in both cell lines (left panel: ARPE-19 cells; right panel: BALB-3T3 cells). Notably, for BALB-3T3 cells, some samples have not been tested (see Table 2).

Figure 3

Constellation plot for both cell lines (left panel: ARPE-19 cells; right panel: BALB-3T3 cells). Constellation plot arranges each PFC sample according to their similarity herein because of both H-content and relative cytotoxicity. The length of the lines between points approximates the distance among clusters. Notably, for BALB-3T3 cells, some samples have not been tested (see Table 2).