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

Laia Tolosa,Bob van der Water,Sylvia E. Escher,M. A. Miranda,Teresa Martínez-Sena,Bas ter Braak,José V. Castell,Erika Moro,Johannes P. Schimming,Barbara M. A. van Vugt-Lussenburg

doi:10.1007/s00204-021-03036-w

Abstract

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.

Highlights

The assessment of the toxicity of chemicals in in vitro assays is frequently hampered by problems related to solubility, volatility or instability
Assessing toxicity of phenolic compounds is of interest because they are widely used by industry, are present in many trade products and as environmental pollutants (Downs and Wills 2020; Kahru et al 2002; Zapor 2004)
To make the in vitro study of the toxicity of phenols feasible and reliable, 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 that were placed on the top of multi-well plates replacing the conventional plastic lid, to minimize diffusion and oxidation

Summary

Introduction

The assessment of the toxicity of chemicals in in vitro assays is frequently hampered by problems related to solubility, volatility or instability. Assessing toxicity of phenolic compounds is of interest because they are widely used by industry, are present in many trade products and as environmental pollutants (Downs and Wills 2020; Kahru et al 2002; Zapor 2004). Phenols are difficult to handle because of degradation, oxidation (Yamamura 2009) and volatility that might result in artifactual results as well in contamination of neighbouring wells (Piersma et al 2013). Archives of Toxicology (2021) 95:2109–2121 phenols and potential losses of tested compounds via degradation and/or diffusion has been seldom addressed in in vitro studies (Pradeep et al 2017; Zhao et al 2020). Efforts to develop new methods for evaluating the safety of volatile chemicals has been demanded by the new REACH regulation (EUREACH 2020)

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Conclusion