Toxicological Assessment of Newly Expressed Proteins (NEPs) in Genetically Modified (GM) Plants


  • Jason Roper Corteva Agriscience™, Johnston, IA
  • Elizabeth A. Lipscomb BASF Corporation, Research Triangle Park, NC
  • Jay S. Petrick Bayer Crop Science, Chesterfield, MO
  • Rakesh Ranjan BASF Corporation, Research Triangle Park, NC
  • Alaina Sauve-Ciencewicki Syngenta Crop Protection, LLC., Research Triangle Park, NC
  • Laurie Goodwin CropLife International, Washington, DC



genetically modified, toxicological assessment, food and feed, hazard, exposure, risk, core studies, supplementary studies


This paper details the weight of evidence (WOE) and stepwise approaches used to assess the food and feed safety of newly expressed proteins (NEPs) in genetically modified (GM) plants, based on previously reported principles. The WOE approach is critical, as in a vast majority of cases no single assay or biochemical characteristic can identify a protein as a hazard. A stepwise approach is recommended to evaluate the safety of NEPs taking the totality of information into account. Potential triggers for the need for supplementary toxicology studies are discussed, and an alternative in vitro method for the acute toxicology study is proposed.

doi: 10.21423/jrs-v09i1roper


Bushey, D. F., Bannon, G. A., Delaney, B. F., Graser, G., Hefford, M., Jiang, X., Lee, T. C., Madduri, K. M., Pariza, M., Privalle, L. S., Ranjan, R., Saab-Rincon, G., Schafer, B. W., Thelen, J. J., Zhang, J. X. Q., & Harper, M. S. (2014). Characteristics and safety assessment of intractable proteins in genetically modified crops. Regulatory Toxicology and Pharmacology, 69(2), 154-170.

Cao, S., He, X., Xu, W., Ran, W., Liang, L., Luo, Y., Yuan, Y., Zhang, N., Zhou, X., & Huang, K. (2010). Safety assessment of Cry1C protein from genetically modified rice according to the national standards of PR China for a new food resource. Regulatory Toxicology and Pharmacology, 58(3), 474-481.

Codex Alimentarius Commission. (2009). Foods derived from modern biotechnology, Second edition. Food and Agriculture Organization of the United Nations and World Health Organization, Rome. Retrieved from

Constable, A., Jonas, D., Cockburn, A., Davi, A., Edwards, G., Hepburn, P., Herouet-Guicheney, C., Knowles, M., Moseley, B., Oberdorfer, R., & Samuels, F. (2007). History of safe use as applied to the safety assessment of novel foods and foods derived from genetically modified organisms. Food and Chemical Toxicology, 45(12), 2513-2525.

Deikman, J., Petracek, M., & Heard, J. E. (2012). Drought tolerance through biotechnology: improving translation from the laboratory to farmers' fields. Current Opinion in Biotechnology, 23(2), 243-250.

Delaney, B. (2017). In vitro studies with human intestinal epithelial cell line monolayers for protein hazard characterization. Food and Chemical Toxicology, 110, 425-433.

Delaney, B., Astwood, J. D., Cunny, H., Conn, R. E., Herouet-Guicheney, C., MacIntosh, S., Meyer, L. S., Privalle, L., Gao, Y., Mattsson, J., & Levine, M. (2008). Evaluation of protein safety in the context of agricultural biotechnology. Food and Chemical Toxicology, 46(Suppl 2), S71-S97.

Eaton, A. D., Zimmermann, C., Delaney, B., & Hurley, B. P. (2017). Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein. Food and Chemical Toxicology, 106, 70-77.

EFSA Panel on Genetically Modified Organisms. (2011). Guidance for risk assessment of food and feed from genetically modified plants. EFSA Journal, 9(5). doi: 10.2903/j.efsa.2011.2150

Erickson, R. H., & Kim, Y. S. (1990). Digestion and absorption of dietary protein. Annual Review of Medicine, 41(1), 133-139.

Farmer, D. R., Edrington, T. C., Kessenich, C. R., Wang, C., & Petrick, J. S. (2017). Improving insect control protein activity for GM crops: A case study demonstrating that increased target insect potency can be achieved without impacting mammalian safety. Regulatory Toxicology and Pharmacology, 89, 155-164.

Gardner, M. L. G. (1988). Gastrointestinal Absorption of Intact Proteins. Annual Review of Nutrition, 8(1), 329-350.

Goldberg, M., & Gomez-Orellana, I. (2003). Challenges for the oral delivery of macromolecules. Nature Reviews Drug Discovery, 2(4), 289-295.

Habig, J. W., Rowland, A., Pence, M. G., & Zhong, C. X. (2018). Food safety evaluation for R-proteins introduced by biotechnology: A case study of VNT1 in late blight protected potatoes. Regulatory Toxicology and Pharmacology, 95, 66-74.

Hamman, J. H., Enslin, G. M., & Kotze, A. F. (2005). Oral delivery of peptide drugs. BioDrugs, 19(3), 165-177.

Hammond, B., Kough, J., Herouet-Guicheney, C., & Jez, J. M. (2013). Toxicological evaluation of proteins introduced into food crops. Critical Reviews in Toxicology, 43(sup2), 25-42.

Hammond, B. G., & Jez, J. M. (2011). Impact of food processing on the safety assessment for proteins introduced into biotechnology-derived soybean and corn crops. Food and Chemical Toxicology, 49(4), 711-721.

Hefferon, K. L. (2015). Nutritionally enhanced food crops; progress and perspectives. International Journal of Molecular Sciences, 16(2), 3895-3914.

Hendriks, H. G., Kik, M. J., Koninkx, J. F., Van den Ingh, T. S., & Mouwen, J. M. (1991). Binding of kidney bean (Phaseolus vulgaris) isolectins to differentiated human colon carcinoma Caco-2 cells and their effect on cellular metabolism. Gut, 32(2), 196-201.

Hurley, B. P., Eaton, A. D., Zimmermann, C., & Delaney, B. (2016). Polarized monolayer cultures of human intestinal epithelial cell lines exposed to intractable proteins - In vitro hazard identification studies. Food and Chemical Toxicology, 98(Part B), 262-268.

Hurley, B. P., Pirzai, W., Eaton, A. D., Harper, M., Roper, J., Zimmermann, C., Ladics, G. S., Layton, R. J., & Delaney, B. (2016). An experimental platform using human intestinal epithelial cell lines to differentiate between hazardous and non-hazardous proteins. Food and Chemical Toxicology, 92, 75-87.

Ishiguro, M., Nakashima, H., Tanabe, S., & Sakakibara, R. (1992). Interaction of Toxic Lectin Ricin with Epithelial Cells of Rat Small Intesting in Vitro. Chemical and Pharmaceutical Bulletin, 40(2), 441-445.

Juberg, D. R., Herman, R. A., Thomas, J., Brooks, K. J., & Delaney, B. (2009). Acute and repeated dose (28 day) mouse oral toxicology studies with Cry34Ab1 and Cry35Ab1 Bt proteins used in coleopteran resistant DAS-59122-7 corn. Regulatory Toxicology and Pharmacology, 54, 154-163.

Kier, L. D., & Petrick, J. S. (2008). Safety assessment considerations for food and feed derived from plants with genetic modifications that modulate endogenous gene expression and pathways. Food and Chemical Toxicology, 46(8), 2591-2605.

Lafont, J., Rouanet, J. M., Gabrion, J., Assouad, J. L., Infante, J. L. Z., & Besancon, P. (1988). Duodenal toxicity of dietary Phaseolus vulgaris lectins in the rat: an integrative assay. Digestion, 41(2), 83-93.

Lodish, H. F., Berk, A., Kaiser, C. A., Krieger, M., Scott, M. P., Bretscher, A., Ploegh, H. L., & Matsudaira, P. (2008). Molecular Cell Biology, Sixth Edition. W.H. Freeman and Company.

Markell, L. K., Wezalis, S. M., Roper, J. M., Zimmermann, C., & Delaney, B. (2017). Incorporation of in vitro digestive enzymes in an intestinal epithelial cell line model for protein hazard identification. Toxicology in Vitro, 44, 85-93.

Mathesius, C., Barnett, J. F., Jr., Cressman, R. F., Ding, J., Carpenter, C., Ladics, G. S., Schmidt, J., Layton, R. J., Zhang, J. X. Q., Appenzeller, L. M., Carlson, G., Ballou, S., & Delaney, B. (2009). Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans. Regulatory Toxicology and Pharmacology, 55(3), 309-320.

Mendelsohn, M., Kough, J., Vaituzis, Z., & Matthews, K. (2003). Are Bt crops safe? Nature Biotechnology, 21(9), 1003-1009.

Moar, W. J., Evans, A. J., Kessenich, C. R., Baum, J. A., Bowen, D. J., Edrington, T. C., Haas, J. A., Kouadio, J. L. K., Roberts, J. K., Silvanovich, A., Yin, Y., Weiner, B. E., Glenn, K. C., & Odegaard, M. L. (2017). The sequence, structural, and functional diversity within a protein family and implications for specificity and safety: The case for ETX_MTX2 insecticidal proteins. Journal of Invertebrate Pathology, 142, 50-59.

O'Hagan, D. T., Palin, K. J., & Davis, S. S. (1987). Intestinal absorption of proteins and macromolecules and the immunological response. Critical Reviews in Therapeutic Drug Carrier Systems, 4(3), 197-220.

Organisation for Economic Co-operation and Development. (2001). OECD Guideline for Testing of Chemicals: Acute Oral Toxicity - Up-and-Down Procedure, 425. Retrieved July 2019 from

Rossi, M. A., Mancini Filho, J., & Lajolo, F. M. (1984). Jejunal ultrastructural changes induced by kidney bean (Phaseolus vulgaris) lectins in rats. British Journal of Experimental Pathology, 65(1), 117-123.

Rothstein, S. J., Bi, Y-M., Coneva, V., Han, M., & Good, A. (2014). The challenges of commercializing second-generation transgenic crop traits necessitate the development of international public sector research infrastructure. Journal of Experimental Botany, 65(19), 5673-5682.

Schneeberger, E. E., & Lynch, R. D. (2004). The tight junction: a multifunctional complex. American Journal of Physiology - Cell Physiology, 286(6), C1213-C1228.

Shah, R. B., Ahsan, F., & Khan, M. A. (2002). Oral Delivery of Proteins: Progress and Prognostication. Critical Reviews™ in Therapeutic Drug Carrier Systems, 19(2).

Sjoblad, R. D., McClintock, J. T., & Engler, R. (1992). Toxicological considerations for protein components of biological pesticide products. Regulatory Toxicology and Pharmacology, 15(1), 3-9.

Stagg, N. J., Thomas, J., Herman, R. A., & Juberg, D. R. (2012). Acute and 28-day repeated dose toxicology studies in mice with aryloxyalkanoate dioxygenase (AAD-1) protein expressed in 2,4-D tolerant DAS-40278-9 maize. Regulatory Toxicology and Pharmacology, 62(2), 363-370.

Tornqvist, E., Annas, A., Granath, B., Jalkesten, E., Cotgreave, I., & Oberg, M. (2014). Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing. PloS One, 9(7), e101638.

U.S. Environmental Protection Agency. (2002). Health Effects Test Guidelines: OPPTS 870.1100 Acute Oral Toxicity. Retrieved July 2019 from

U.S. Food and Drug Administration (2016). Generally Recognized as Safe (GRAS). Retrieved July 2019 from

Vasconcelos, I. M., & Oliveira, J. T. A. (2004). Antinutritional properties of plant lectins. Toxicon, 44(4), 385-403.

Weinman, M. D., Allan, C. H., Trier, J. S., & Hagen, S. J. (1989). Repair of microvilli in the rat small intestine after damage with lectins contained in the red kidney bean. Gastroenterology, 97(5), 1193-1204.

Xu, W., Cao, S., He, X., Luo, Y., Guo, X., Yuan, Y., & Huang, K. (2009). Safety assessment of Cry1Ab/Ac fusion protein. Food and Chemical Toxicology, 47(7), 1459-1465.

Yoder, J. M., Aslam, R. U., & Mantis, N. J. (2007). Evidence for widespread epithelial damage and coincident production of monocyte chemotactic protein 1 in a murine model of intestinal ricin intoxication. Infection and Immunity, 75(4), 1745-1750.




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