Sublethal Endpoints in Non-target Organism Testing for Insect-Active GE Crops

Abstract

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis (Bt) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non-Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms.

RNAi for Western Corn Rootworm Management: Lessons Learned, Challenges, and Future Directions

“…The most common endpoint or effect evaluated is mortality, as it is unambiguous, easy to measure, and correlates with adverse effects to field populations. However, sublethal endpoints such as weight and developmental timing have also been used as additional endpoints in studies evaluating the risk of Bt crops (reviewed by Roberts et al [172]).…”

“…The use of RNAi as a new MOA to confer a particular desired phenotype in a genetically modified crop has increased over the past decade [154,174]. Given the flexibility and robustness of the existing ERA framework described above, this structure is well suited to characterize the environmental safety of transgenic crops expressing dsRNA [172]. To briefly illustrate that process, we consider a dsRNA expressed in maize intended to control WCR.…”

“…The effects observed against the target pest, WCR, are informative for both the duration of laboratory studies and the endpoints that should be evaluated in those studies. Traditionally, mortality is the primary endpoint in risk assessment hazard studies with a threshold of at least 50% effect to trigger higher tier tests [168,172]. This is based on the notion that <50% effect at test concentrations in laboratory studies that exceed environmental concentrations are unlikely to result in adverse effects under field conditions.…”

Assessing the Risks of Topically Applied dsRNA-Based Products to Non-target Arthropods

“…The endpoints to be recorded (lethal and sublethal) need to be selected based on the organism under investigation (and the reliability of the test system) and the gene that is targeted. While lethality is an obvious endpoint to be chosen, the consideration of sublethal endpoints such as growth or development time is recommended (Roberts et al, 2020). First, they may hint to unexpected off-target effects, second, they may cover for the fact that dsRNA is generally slow acting (Baum and Roberts, 2014) and that the process is typically not reaching 100% gene suppression (e.g., Bolognesi et al, 2012;Rangasamy and Siegfried, 2012), and third, they might address the fact that RNAi effects can be transgenerational, i.e., also affecting subsequent generations (Abdellatef et al, 2015).…”

“…First, they may hint to unexpected off-target effects, second, they may cover for the fact that dsRNA is generally slow acting (Baum and Roberts, 2014) and that the process is typically not reaching 100% gene suppression (e.g., Bolognesi et al, 2012;Rangasamy and Siegfried, 2012), and third, they might address the fact that RNAi effects can be transgenerational, i.e., also affecting subsequent generations (Abdellatef et al, 2015). Sublethal endpoints are typically also recorded in the testing of chemical pesticides (e.g., Candolfi et al, 2000) and Bt proteins (De Schrijver et al, 2016;Roberts et al, 2020) even though mortality is the primary endpoint and often the results from testing sublethal endpoints are not reported in regulatory summaries. In any case, it is important to set decisionmaking criteria for every endpoint that is recorded.…”