The bioavailability of volatile organic compounds (VOCs) in a given situation often remains challenging to assess, and the lack of standard methods for introducing VOCs into in vitro bioassays can lead to poorly defined bioavailable concentrations. As a result, in vitro assays normally conducted in wells of cell culture plates for risk assessment of volatile and hydrophobic organic chemicals (VHOCs) have always faced significant experimental difficulties due to high volatility and high hydrophobicity. This compromises the true exposure concentration by: (i) causing the amount of test substances in the test medium to decline, (ii) limits the quality of toxicological responses and their extrapolation, and thus, can lead to interpretational errors. In the research herein, a dosing method was developed to assess the bioavailability of benzene (that served as a model for VHOCs) in aqueous tests and to better characterize exposure estimates for an improved risk assessment during in vitro biotests. This study hypothesizes that (1) benzene bioavailability to intestinal porcine enterocyte cell line (IPEC-1 cells) can be partially explained by phase partitioning, as measured by freely dissolved concentration that drives the diffusive uptake into the cell membranes, (2) benzene equilibrium partitioning between the donor and the cell membranes is dependent upon energetic state of the chemical concentration in the partitioning donor, which describes its chemical activity. Silicone polydimethylsiloxane (PDMS) was used as the partitioning donor for passive dosing in transwell plates. The buffering capacity of the donor compensates for routine loss against depletion processes during the toxicity tests, resulting in stable exposure concentrations of benzene freely available to cells at relatively constant chemical activity. For IPEC-1 cells in the passive dosing tests, the median effective concentration (EC50) was 4.82 mg/L. The obtained median effective activity (Ea50) value is within the chemical activity range (0.01– 0.1) for baseline toxicity of several hydrophobic chemicals reported in the literature. Cell inhibition ranged from 9.6 ± 2% to 97.7 ± 0.8% for freely dissolved concentrations of benzene, which ranged from 0.6 to 5.4 mg/L after 24 h exposure. The spiking tests result in an ECSpike-50 projected to be greater than 5.4 mg/L, (highest spiked concentration) and reduced test sensitivity of benzene to IPEC-1 cells. This study introduces a new effective approach to passive dosing and demonstrates the utility of passive dosing over solvent spiking for in vitro toxicity testing of hydrophobic chemical (log Kow ˂ 4.6) with high volatility. This has fundamental implications for a better understanding of the interactions between VHOCs exposure to humans and the toxic effects on the human intestine to help set remediation objectives and further the improve future risk assessment and standard setting for VHOCs
