Hazard assessment of insecticidal proteins representing different structural classes using an in vitro experimental platform of human intestinal epithelial monolayers.

Hazard assessment of proteins using human-derived intestinal epithelial cell (IEC) lines has been investigated previously. The purpose of this work was to a) reduce occurrences of false positives by including a non-hazardous protein (BSA) negative control while redesigning statistical analyses; and b) consider the potential of the IEC assay as a component of a weight of evidence approach in the hazard assessment of four different structural/functional classes of insecticidal proteins. The protein classes evaluated included: Mpf (Mpf2Ba1); Cyt (IPD084Aa); Cry (IP3-H9); and uncharacterized (IPD072Aa). Using BSA as the negative control comparator paired with a statistical method correcting for false positives, it was determined that three of the four proteins evaluated did not reveal any indications of hazard. Statistically significant effects on barrier integrity and cytotoxicity endpoints were observed for intact (non-digested) IPD072Aa protein, however, no adverse effects were observed in acute and repeated dose oral toxicity studies with IPD072Aa. Use of the IEC assay featuring refinements to better distinguish between false negatives and effects that are significant and reproducible may serve as a useful component within a weight of evidence approach to inform the level of downstream in vivo testing warranted to identify potentially hazardous proteins.