Impact of ethanol as a vehicle for water-insoluble pollutants in BEAS-2B cell toxicity assays

Abstract

In vitro human cell models are the gold standard for toxicological screening of environmental pollutants, allowing precise profiling of cellular responses. Pollutants with limited water solubility require carrier vehicles for uniform exposure. Ethanol, a commonly used vehicle, is typically maintained at 0.05–1.0% (v/v) to minimize toxicity. However, definitive no-observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs) for ethanol in non-tumorigenic human bronchial epithelial (BEAS-2B) cells, prevalent in inhalation studies, have not been established. Researchers thus apply a range of ethanol concentrations derived from diverse cell lines, increasing the risk of vehicle interference. This study evaluated ethanol as a cosolvent vehicle for four emerging high-flashpoint hydrocarbon (HFHC) dry cleaning solvents in BEAS-2B cells. HFHC solvents were solubilized 1:1 in 100% ethanol, then diluted in bronchial epithelial cell growth basal medium to final concentrations of 0.05%, 0.25%, 0.5%, and 2.5% (v/v). Vehicle, positive, and negative controls isolated ethanol-specific cytotoxic effects. Cytotoxicity was assessed via cellular viability (MTS assay) at 24 and 48 h, and lactate dehydrogenase (LDH) and interleukin-8 (IL-8) release after 24 h. Ethanol drove viability loss at ≥0.5% (24 h) and ≥0.25% (48 h), induced inflammation at concentrations ≥0.05%, and minimally impacted membrane integrity. Most HFHC solvents showed minimal effects beyond ethanol alone, except one HFHC, Intense, causing significant membrane disruption and cytotoxicity even at low doses (0.05–0.25%). Practical ethanol noninterference thresholds recommended are ≤0.5% for 24-hour assays, ≤0.25% for 48-hour viability, and ≤0.05% for inflammatory endpoints, establishing critical guidelines for ethanol use in BEAS-2B assays.