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

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.

Toxic effects of trace phenol/guanidine isothiocyanate (P/GI) on cells cultured nearby in covered 96-well plates

“…However, it is possible that such changes occur in select processes even at large distances. Given the volatile nature of P/GI [ 2 ], fumes from trace P/GI left over after lysing cells at 1 collection point may affect other cells on the same culture plate at a later collection point. To avoid the confounding effects of even minute quantities of P/GI, laboratories should take these observations into account when designing time-course experiments in tissue culture plates.…”

“…In controlled time-course experiments in multi-well tissue culture plates, replicate cultures can be treated with a stimulus at one time and harvested at various times afterwards using P/GI. Phenolic compounds [ 2 ] and guanidine compounds [ 3 ] are cytotoxic, but the incidental effects of trace amounts of these volatile substances, either separately or in combination, on adjacent cell culture wells (as opposed to direct exposure in culture medium) has not been investigated.…”

Ready for regulatory use: NAMs and NGRA for chemical safety assurance

“…Various groups have reviewed potentially difficult-to-test materials that would perhaps not normally be suitable for testing under standard in vitro conditions and have considered ways in which this can be accounted for when interpreting the data. These have included volatile phenols, highly oxidizable and reactive compounds and nano materials amongst others (Tolosa et al, 2021;Drasler et al, 2017;Proenca et al, 2021) and have highlighted the need to increase the understanding of identity and quantity of a compound exposed in in vitro systems; accurately interpreting the dose at which an effect occurs. The documentation and quantification of such uncertainties will be key to increasing regulatory acceptance of NAMs.…”

Efficient Catalytic Degradation of Phenol with Phthalocyanine-Immobilized Reduced Graphene–Bacterial Cellulose Nanocomposite

“…Phenol and phenol derivatives are widely used in a series of fields such as petrochemicals, pharmaceuticals, textile manufacturing, wood products, steel foundries, etc. The resulting phenolic compound-containing effluents are classified as one of the highest priority types of industrial pollutants due to their toxicity to aquatic organisms and human beings, even at low concentrations [ 1 , 2 ]. The increasing concern regarding their environmental and health risks prompted the implementation of effective treatment techniques that are capable of dealing with phenolic compounds.…”