The search for a truly reliable cytotoxicity assay on perfluorocarbon liquids (PFCLs) is becoming one of the “hottest” topics in vitreoretinal surgery.
Since the very unfortunate events occurred during the recent years using perfluorocarbon liquids endotamponades, surgeons want to be reassured that these incidents will not reoccur. With this aim, PFCL manufacturers are seriously committed to do their best to provide surgeons with strong evidences on their product safety.
It is now ascertained that completely fluorinated, unreactive PFCL products such as PFO (perfluoro-n-octane) and PFD (perfluorodecalin) are not toxic by themselves; therefore, all research efforts are now focusing on the search of the best design attributes to detect or, more properly, to exclude the presence of impurities that might lead to ocular toxicity.
Nevertheless, the latest literature clearly demonstrates that this topic is still matter of a great debate, and that an overall agreement in this field is lacking.
Currently, two different approaches are in the spotlight: the H-value determination and the in vitro cytotoxicity tests using direct contact methods.
Menz’s group of research from the Pharmpur Gmbh, while highlighting the clinical relevance of the so-called H-value, defined as the residual amount of reactive incompletely fluorinated compounds in PFCLs, proposed that “The H-value is a measure for reactive and underfluorinated impurities that cause toxicity of PFCLs and should be incorporated in each endotamponade specification with a limit of 10 ppm to prove the effectiveness of the ultra-purification required and ensure a safe product”. The proposal was based on the “evidence that […] incompletely fluorinated by-products that inevitably arise during production and are dissolved in PFCL can, even in minutes quantities, lead occasionally to severe toxic reactions”, and that “the reactivity of incompletely fluorinated compounds and their toxicity are closely related”.
The doubts arisen on the H-value determination method included, among other things, its ability to detect only specific underfluorinated compounds of the form RFRH (having reactive C-H bonds) while leaving undetected other potentially toxic chemical compounds – which were identified in the “unsafe” PFCL used in Spain – such as acids, alcohols, and benzene derivatives; in addition, it was recently shown that using an innovative NMR technology “failed to demonstrate the correlation between the H-content and in vitro cytotoxicity test in ARPE-19 and BALB/3T3 cell lines”. Nevertheless, it must be recognized that the Menz’s group of research admitted that “the article is the first contribution in a planned series of publications aimed at providing tools suitable for ensuring patient safety in the ophthalmological application of perfluoro-carbon liquids (PFCL)”.
THE CITOTOXICITY TEST
A growing interest is gaining the in vitro cytotoxicity test by direct contact method on human retinal pigment epithelial cell line (ARPE-19) according to ISO 10993-5 recommendations.
It should be noted that the direct contact method is, among all the methodologies recommended by the ISO 10993-5 standard, highly sensitive and able to detect even weak cytotoxicity. In addition, as stated by the ISO standard, the application of the PFCL directly on human retinal pigment epithelial cell line (ARPE-19) is, as much as possible, similar to the clinical situation in which the product will be used. Instead, other tests included in the ISO 10993-5 such as the extract method do not obey to this requirement and are much less sensitive for testing volatile and immiscible in water substances as the PFCLs: indeed, in the past the use of the extract method led to falsely negative results, with the well-known bad clinical outcomes in Spanish patients.
It is certainly for all the above-mentioned reasons that two groups of research, the Srivastava’s research group from the IOBA Institute (Instituto Universitario de OftalmobiologÍa Aplicada, Spain), and the team of researchers from Alchilife Srl (Italy), in collaboration with Mario Romano, PhD, from the Humanitas University of Milano (Italy), have dedicated a great effort in tuning up reliable direct contact methods. In fact, this approach requires few, yet very important, technical steps that are strictly related to the physical-chemical specifications of PFCLs and crucial for validating the direct contact method. If, from one side, the high-density properties of these compounds help avoiding any sample evaporation or loss of contact with the cells, from the other one they may lead to insufficient contact area and contact time between the sample and the cell layer, cell mortality induced by sample heaviness and inappropriate cell sensibility. Thus, a careful assessment of all risks of possible direct contact test failure must be performed.
All these requirements were fulfilled by the two research groups, and each of them proposed their validated method:
- In a paper published on IOVS (2018), Srivastava et al. commented that their study on ARPE-19 and mouse fibroblast L929 cell lines “suggests applying this new method to avoid occurrence of such cases of blindness”;
- In his presentation at the 18th Euretina Congress in Vienna, M. Romano affirmed that “The direct contact cytotoxicity test according to ISO 10993-5 is a suitable method to detect the cytotoxicity of PFCLs that was validated using both a quantitative and a qualitative approach in ARPE-19 and BALB/3T3 cell.” This presentation is of particular relevance for Alchimia, since the described method is the one that has always been used to test their PFCLs, as shown on the finished product labels.
Let us conclude with these few considerations:
- Although a uniform evaluation method or cytotoxicity test evaluation has not been established so far, some truly reliable cytotoxicity test methods are becoming available in literature, which might help overcoming the surgeon safety concerns about the use of PFCLs in vitreoretinal surgery;
- A more efficient control by the European Union Authorities is welcome, yet lacking;
- Even the most reliable cytotoxicity tests must be accompanied by very thorough and most accurate chemical-physical tests to guarantee the highest purity of PFCLs.
- Pastor JC et al. Acute retinal damage after using a toxic perfluorooctane for vitreo-retinal surgery. Retina 2017;1440–1151
- Menz DH, et al. How to ward off retinal toxicity of perfluorooctane and other perfluorocarbon liquids?
Invest Ophthalmol Vis Sci. 2018; 59:4841–4846
- Srivastava GK et al. Chemical considerations regarding the H-value methodology and its relation with toxicity determination.
Invest Ophtahalmol Vis Sci 2019; 60:3363-3364
- Ruzza P et al. H‐content is not predictive of perfluorocarbon ocular endotamponade cytotoxicity in vitro.
ACS Omega 2019 https://doi.org/10.1021/acsomega.9b01793
- Menz DH et al. Author Response: Chemical Considerations Regarding the H-Value Methodology and Its Relation With Toxicity Determination.
Invest Ophthalmol Vis Sci 2019;60(10):3365-3367
- ISO 10993-5, 2009. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity.
- Romano M. et al. Evaluation of cytotoxicity of perfluorocarbons for intraocular use by cytotoxicity test in vitro in cell lines and human donor retina ex vivo. Trans Vis Sci Technol 2019, in press.
- Srivastava GK et al. Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation.
Sci Rep 2018;8(1):1425