Evaluation of an in vitro experimental platform of human polarized intestinal epithelial monolayers for the hazard assessment of insecticidal proteins.

Previous work demonstrated the utility of using human-derived intestinal epithelial cell (IEC) lines cultured as polarized monolayers on Transwell® filters to differentiate between hazardous and non-hazardous proteins. The current study seeks to further resolve appropriate concentrations for evaluating proteins of unknown hazard potential using the IEC experimental platform and leverages these parameters for evaluating the potential toxicity of insecticidal proteins characteristic of those expressed in genetically modified (GM) agricultural biotechnology crops. To establish optimal test protein concentrations, effects of several known hazardous (C. perfringens epsilon toxin, Listeriolysin O, Phaseolus vulgaris erythroagglutinin, E. coli Shiga toxin 1, C. difficile Toxin B and wheat germ agglutinin) and non-hazardous (Ara-h2, ß-lactoglobulin, fibronectin and Rubisco) proteins on IEC barrier integrity and cell viability were evaluated at concentration ranges. Two insecticidal proteins (AfIP-1A and AfIP-1B) were evaluated for effects in the IEC assay, a seven-day insecticidal bioassay, and assessed in a high-dose 14-day acute oral toxicity study in mice. The results obtained from the human in vitro IEC assay were consistent with results obtained from an in vivo acute oral toxicity study, both demonstrating that the combination of AfIP-1A and AfIP-1B do not exhibit any identifiable harmful impacts on mammalian cells.

Protein familiarity is a fundamental but rarely operationalized concept in the safety assessment of genetically modified crops: example of phosphomannose isomerase (PMI).

Fundamental to the safety assessment of genetically modified (GM) crops is the concept of negligible risk for newly expressed proteins for which there is a history of safe use. Although this simple concept has been stated in international and regional guidance for assessing the risk of newly expressed proteins in GM crops, its full implementation by regulatory authorities has been lacking. As a result, safety studies are often repeated at a significant expenditure of resources by developers, study results are repeatedly reviewed by regulators, and animals are sacrificed needlessly to complete redundant animal toxicity studies. This situation is illustrated using the example of the selectable marker phosphomannose isomerase (PMI) for which familiarity has been established. Reviewed is the history of safe use for PMI and predictable results of newly conducted safety studies including bioinformatic comparisons, resistance to digestion, and acute toxicity that were repeated to gain regulatory reapproval of PMI expressed from constructs in recently developed GM maize. As expected, the results of these newly repeated hazard-identification and characterization studies for PMI indicate negligible risk. PMI expressed in recently developed GM crops provides an opportunity to use the concept of familiarity by regulatory authorities to reduce risk-disproportionate regulation of these new events and lessen the resulting waste of both developer and regulator resources, as well as eliminate unnecessary animal testing. This would also correctly imply that familiar proteins like PMI have negligible risk. Together, such modernization of regulations would benefit society through enabling broader and faster access to needed technologies.

Safety assessment of the insecticidal protein IPD079Ea from the fern, Ophioglossum pendulum.

As agricultural biotechnology continues to develop solutions for addressing crop pests through newly expressed proteins from novel source organisms, with different modes or sites of action and/or different spectra of activity, the safety of these proteins will be assessed. The results of hazard-identification and characterization studies for the insecticidal protein IPD079Ea, which is derived from a fern (Ophioglossum pendulum) and active against the maize pest western corn rootworm (Diabrotica virgifera virgifera, Coleoptera: Chrysomelidae) are provided. Collectively these results indicate that IPD079Ea is unlikely to present a hazard to human or animal health and support the safety of genetically modified maize expressing IPD079Ea.

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk.

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

Safety assessment of coleopteran active IPD072Aa protein from Psuedomonas chlororaphis.

The ipd072Aa gene from Pseudomonas chlororaphis encodes the IPD072Aa protein which confers protection against certain coleopteran pests when expressed in genetically modified (GM) plants. A weight of evidence approach was used to assess the safety of the IPD072Aa protein. This approach considered the history of safe use of the source organism and bioinformatic comparison of the protein sequence with known allergenic and toxic proteins. The IPD072Aa protein was assessed for resistance to degradation in the presence of simulated gastric fluid containing pepsin as well as heat stability. There was no hazard identified with the IPD072Aa protein. Furthermore, an acute oral toxicity study found no evidence of adverse effects. Collectively, these studies support the human health safety assessment of the IPD072Aa protein.