In the present study, we confirmed the relevance of well-documented knowledge about reactive underfluorinated impurities being a risk factor for PFCL safety for medical application as a blood substitute
14,15,17 for ophthalmic use. The correlation between the H-value and cytotoxic effects of the individually analyzed batches is overwhelming. In addition, we demonstrated that a multistage ultra-purification process of the cytotoxic batch 061014, which eliminated underfluorinated impurities completely, transformed the batch into a well-tolerable material not triggering any cell-growth inhibition. Via the dilution experiment, our evidence reveals that the cytotoxicity increases gradually in conjunction with a rising H-value.
Beginning in 2013, repeated cases of toxic reactions of perfluorooctane used in vitreoretinal surgeries have been reported. A new series of reports on vision loss was published after use of ala octa in Spain in 2015.
11 The focus of the present work was the investigation of affected ala octa batches, because of the best documentation of the cases and the availability of original samples of the affected batches in sufficient quantity. In addition, samples of identical batches were the basis of the publication of Pastor et al.,
11 which clearly identified the PFO batches used as the trigger for the toxic effects. However, the causal link between the cytotoxic effect and their substance specific properties remained vague. Nearly three decades ago, research on the use of various PFCL for medical use as blood substitutes revealed the content of reactive and underfluorinated impurities as being the most important source for PFCL toxicity. To discover whether this is also relevant to the retinal toxicity observed in Spain, we analyzed the PFO batches reportedly associated with those adverse events. Our test results confirm the cytotoxic effects of the PFO batches involved in the severe adverse events in Spain. This proof is even more valuable because a second independent test method for evaluating material cytotoxicity from a second independent organization confirmed findings of the Pastor et al.
11 group, who were the first to confirm by in-vitro testing the clinically diagnosed toxic reactions. Our results on cell growth inhibition are consistent with the findings of Pastor et al.
11 (see
Table 4). This is an important fact, as Pastor et al.
11 claimed that there is no alternative to their direct contact method using ARPE-19 and porcine neuroretina explants, for which a patent was filed.
13 Furthermore, for the determination of cell growth inhibition the established and recognized sensitive cell line L929 was used for the characterization of the toxic potential to allow a good comparability to a large number of substances already tested to evaluate their cytotoxicity. Biocompatibility studies require an extended test protocol compared to pure cytotoxicity studies. Possible advantages of using retinal cell lines, such as ARPE 19, only become apparent in this context.
11
Our analytical evidence closes the gap where a causal link between the phenomenon “cytotoxicity” and a measurable material property of the notorious PFO batches had been sought. The missing explanation caused—quite understandably—uneasiness among users and regulatory authorities, as there seemed to be some mysterious thing about PFCL, which, since unknown, could not be controlled and use of PFCL in vitreoretinal surgery would have been (if at all) safe only by coincidence. Such fears can now be allayed by the results of the present study, which confirm the requirement established for PFCL use as blood substitute, that they should be practical free of reactive underfluorinated impurities (below 5·10
−5 mol/L cleavable fluoride-ions) and allow for safe use in vitreoretinal surgery.
16 To the best of our knowledge, we are the first to have demonstrated the causal chain—reactive impurities in PFCL → high H-value → cytotoxicity → adverse event caused by toxic reaction during ophthalmological application. One has to keep in mind that these acute toxic reactions are completely different from the previously described and known side effects of long-term applications of PFO/PFCL.
Since the transferability of the toxicological investigations of PFCL for use as blood-substitutes to vitreoretinal PFCL use has now been proven, the importance of determining the H-value cannot be overemphasized.
The root-cause of the retinal toxicity is the reactivity of the impurities. In contrast to the extreme chemical stability of fully fluorinated PFC molecules, the impurities can react with chemical and biological material, or they can be converted to other toxic substances. The determination of the H-value relies on exactly this reactivity of the impurities. To detect all reactive compounds completely, the conditions of the transformation reaction must be very harsh, meaning that any potential toxic impurity will be converted and thereby detected. At the same time, all of the transformations that already occurred in the sample under HF formation are also revealed through fluoride-selective potentiometry.
Any fully fluorinated PFCL, by its inert nature, will pass through the transformation reaction without any change. To exclude any latent risk of toxicity, PFCLs should be practically free of reactive impurities. Applying the method for determining the H-value described here, an H-value not exceeding 10 ppm is equivalent to this stringent criterion, because 10 ppm is the validated, robust limit of this test method. Higher values indicate the presence of reactive underfluorinated compounds in the PFCL material we investigated, and hence the potential risk of material toxicity.
A further conclusion from these studies should be discussed: we acknowledge that the validated limit of 10 ppm for the H-values derived from the detection limit is a sufficient criterion to conclude that a PFCL is not cytotoxic. However, that does not mean that every noncytotoxic PFCL must necessarily have an H-value of ≤10 ppm. As referenced in the introduction, different hydrogen-containing and unsaturated impurities reveal different toxic effects.
15 The fact that our dilution study suggests a steady increase in cytotoxicity with an increasing H-value must not be overinterpreted. In that test, we examined a toxic material with an unchanged impurity profile in combination with an ultra-purified, nontoxic material. On the contrary, the results in
Table 3 prove that each batch has its individual relationship between the H-value and cytotoxicity despite originating from the same raw material.
To elucidate the underlying reasons, we plan to analyze the chemical composition of individual species in the impurity profile and discuss these in a follow-up publication. It is also important to clarify why the determination of the cytotoxicity of PFCL samples revealed reproducibility problems as reported by Pastor et al.
11 A larger variety of PFCL products for vitreoretinal surgery should also be investigated to evaluate their overall quality and the safety of products currently on the market.
In conclusion, the determination of the H-value is an indispensable tool for assessing the suitability of PFCL for ophthalmic use. The H-value not only represents a random product property, it is the key parameter to evaluate the long-term toxicological potential of a PFCL. This means that the H-value test goes far beyond the phenomenological assessment of a given batch, which would only describe its quality as a snapshot, as this is the case, for example, with an evaluation relying solely on a cytotoxicity measurement.
In conclusion, the recent case series of severe side effects and vision loss after PFO use could have been prevented had the H-value parameter already been the standard parameter for assessing the product quality of PFO or any similar PFCL substance, for example, perfluorodecalin. Therefore, the H-value should be established routine as part of the product specification and final release, and surgeons should request it.
Completely purified and characterized PFCL used as an ocular endotamponade are still safe devices. The toxicity described in connection with individual batches was caused by effects from reactive underfluorinated impurities.