Integrated risk assessment reveals low biological activity of RNA-based crop protection formulations across plant and non-target systems

RNA-based crop protection products are considered promising alternatives to conventional pesticides due to their high target specificity and selectivity profile. While current risk assessments mainly focus on sequence-dependent off-target effects, potential sequence-independent effects of RNA molecules and formulation components remain poorly understood. Here, we assessed the potential of naked and formulated RNA molecules to induce unintended biological responses in plants and non-target organisms using complementary plant stress and cellular toxicity assays. Single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), circular RNA (circRNA), and chitosan-based RNA formulations were evaluated for their ability to induce early stress responses in plants and adverse effects in insect cells. Cytosolic Ca²⁺ signaling and reactive oxygen species (ROS) production were monitored in barley and Arabidopsis, while impedance-based phenotypic profiling (ECIS) was used to assess cellular responses in Spodoptera frugiperda (Sf21) cells. Neither naked nor formulated RNA preparations triggered pronounced Ca²⁺ influxes or ROS bursts, indicating an absence of substantial activation of early plant defense signaling. Likewise, RNA formulations caused only minor effects on insect cell attachment and spreading as a measure for cell viability. Observable responses occurred primarily at elevated concentrations of RNA formulations and were formulation-dependent rather than RNA-dependent. Overall, the investigated RNA molecules exhibited low biological activity across the tested non-target systems. Our results highlight the importance of evaluating RNA active ingredients and delivery matrices separately and demonstrate the value of integrated screening approaches for the early hazard assessment of RNA-based crop protection products.