Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays—Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction

Abstract

In vitro assays are normally conducted in plastic multi-well plates open to exchange with the ambient air. The concentration of test substances freely available to cells is often not known, can change over time, and is difficult to measure in the small volumes in microplates. However, even a well-characterized toxicological response is of limited value if it cannot be linked to a welldefined exposure level. The aim of this study was to develop and apply an approach for determining time resolved freely dissolved concentrations of semi-volatile and hydrophobic organic chemicals (SVHOCs) in in vitro assays: (1) free fractions were measured by a new medium dilution method and (2) time-resolved loss curves were obtained by measurements of total concentrations in 96-well plates during incubations at 37°C. Headspace solid-phase microextraction was used as an analytical technique for 24 model chemicals spanning 6 chemical groups and 4-5 orders of magnitude in K ow and K aw. Free fractions were above 30% for chemicals with log K ow < 3.5 and then decreased with increasing log K ow. Medium concentrations declined significantly (>50%) within 24 hours of incubation for all 20 chemicals having log K ow > 4 or log K aw >-3.5 in serum free medium. Losses of chemicals were lower for medium containing 10% fetal bovine serum, most significantly for chemicals with log K ow > 4. High crossover to neighboring wells was observed also below log K ow of 4 and log K aw of-3.5. Sealing the well plates had limited effect on the losses, but clearly reduced crossover. The high losses and crossover of most tested chemicals question the suitability of multi-well plates for in vitro testing of SVHOCs, and call for (1) test systems that minimize losses, (2) methods to control in vitro exposure, (3) analytical confirmation of exposure and (4) exposure control and confirmation being included in good in vitro reporting standards.

Baseline Toxicity and Volatility Cutoff in Reporter Gene Assays Used for High-Throughput Screening

“…We did not attempt to measure the cytotoxicity of the remaining four chemicals with log K medium/air < 2, they were only included in the thermodynamic analysis as reference. Note that Birch et al included a substantially higher number of such more volatile chemicals in their study of test substance losses and crossover in 96-well plates.…”

“…While solid-phase microextration methods have been developed 24 for this purpose and a practical workflow has been demonstrated using 96-well plates and reporter gene bioassays, 5 any measurement would interfere with the practical bioassay workflow of a typical cell-based bioassay. In an accompanying study, Birch et al 25 have measured the losses and crossover of 24 volatile and semivolatile chemicals from 3 different cell culture media in 96-well plates without cells. Both approaches The column "HTS setup?"…”

Bioanalytical Tools in Water Quality Assessment

“…Loss to air is a problem in well-plate format that is often underestimated. This process might not only lead to loss of chemicals but semi-volatile chemicals can even cross over and contaminate neighbouring wells (Birch et al, 2019). Only chemicals with a medium-air partition constant K medium/air .10 4 are fully retained during 24 hours of exposure at 37°C (Escher et al, 2019).…”

A common contaminant shifts impacts of climate change on a plant-microbe mutualism: effects of temperature, CO2 and leachate from tire wear particles

“…We crossed this design with three levels of tire leachate (96 unique temperature and CO 2 conditions × 3 = 288 treatments) and treated an entire plate with a particular level (0 g/L = none, 5 g/L, or 10 g/L, see above) to prevent cross-contamination between treatments (Birch et al, 2019). We expect that many components of tire leachate could be volatile (U.S. EPA CDC/ATSDR, 2019), yet we require gas exchange for CO 2 treatments and living organisms.…”