The in vitro assessment of the toxicity of volatile, oxidisable, redox-cycling compounds: phenols as an example

Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC–MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.
The authors wish to acknowledge the support of the European Union’s Horizon 2020 research and innovation 493 program under Grant Agreement No 681002 (EU-ToxRisk). We are indebted to CIBEREHD (ISCIII), for supporting our research program on hepatotoxicity of xenobiotics. L.T. was supported by the Miguel 494 Servet I Program financed by the Institute of Health Carlos III (Plan Estatal de I+D+i 2013-2016) 495 and co-financed by the European Regional Development Fund "A way to achieve Europe" (FEDER) 496 through grant CP16/00097. TMS acknowledges her personal predoctoral grant (FI18/00260, 497 ISCIII, Spain).