Haloacetonitrile stability in cell culture media used in vitro toxicological studies

Haloacetonitriles (HANs) are an emerging class of nitrogenous disinfection by-products (DBPs) formed in disinfected drinking water and have been reported to be more cyto- and genotoxic than the regulated DBPs. HANs are also known to hydrolyze under neutral pH and normal room temperature. However, the stability of HANs has not been well characterized in DBP toxicological assessments. Most toxicological assessments expose DBPs up to several days which may result in a mixture of HANs and degradation products that might have underestimated HAN toxicity. In this study, HANs stability was characterized in 1) a buffer solution in sealed vials, 2) cell culture media (CCM) in sealed vials, and 3) CCM in 96 sealed well plates with 5% CO₂. Solutions were incubated at 37 °C for 3 days. MonoHANs were found to be stable in buffer and CCM except when HANs were incubated in CCM in plates where they could possibly be affected by volatilization and photodegradation during sample handling. However, di- and tri- HANs degraded between 70 and 100% in both buffer solution and CCM. They were also found to be less stable in CCM than in buffer solution possibly from HANs reacting with nucleophiles present in CCM (i.e., amino acids). Identified degradation products include corresponding haloacetamides and haloacetic acids for buffer solutions and only haloacetic acids and an unknown brominated compound for CCM. Results of this study suggests that reported toxicity values might have been underestimated and should consider changing CCM and DBP on a daily basis for a more accurate toxicity measurement.