Biotechnology Consultation - Note to File
BNF No. 000140
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Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000140
February 4, 2014
DAS-81419-2, insect-resistant soybean
Soybean, Glycine max, lepidopteran pest resistance, Cry1F, Cry1Ac, Bacillus thuringiensis, phosphinothricin N-acetyltransferase, PAT, Streptomyces viridochromogenes, glufosinate-ammonium, OECD Unique Identifier DAS-81419-2, Dow AgroSciences LLC
This document summarizes our evaluation of biotechnology notification file (BNF) No. 000140. In a submission dated October 15, 2012, Dow AgroSciences LLC (Dow) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of its genetically engineered insect-resistant soybean, transformation event DAS-81419-2 (hereafter referred to as DAS-81419-2 soybean). Dow provided additional information on February 28, 2013. FDA evaluated the information in Dow’s submissions to ensure that regulatory and safety issues regarding human food or animal feed from the new plant variety have been resolved prior to commercial distribution.
In our evaluation, we considered all information provided by the notifier as well as publicly available information and information in the agency’s files. Here we discuss the outcome of the consultation, but do not intend to restate the information provided in the final consultation in its entirety.
The intended effect of the modification in DAS-81419-2 soybean is to confer resistance to several lepidopteran pests of soybean. Dow introduced two genes, where both sequences were derived from Bacillus thuringiensis: (1) a synthetic cry1Fv3 gene composed of sequences of cry1Fa2 from subsp. aizawai, cry1Ca3 from subsp. aizawai, and cry1Ab1 from subsp. berliner and (2) a synthetic cry1Ac(synpro) gene composed of sequences of cry1Ac1 from subsp. kurstaki, cry1Ca3 from subsp. aizawai, and cry1Ab1 from subsp. berliner. The proteins encoded by the cry1Fv3 and cry1Ac(synpro) genes – Cry1F and Cry1Ac, respectively – are toxic to lepidopteran pests including soybean looper, velvet bean caterpillar, fall armyworm, and tobacco budworm.
Dow also introduced the pat gene from Streptomyces viridochromogenes for use as a selectable marker during DAS-81419-2 soybean development. The pat gene encodes phosphinothricin N-acetyltransferase (PAT), which acetylates and thereby inactivates L-phosphinothricin (the active form of glufosinate-ammonium). Growth of plant cells in the presence of the herbicide permitted selection of successful transformants.
The purpose of this evaluation is to assess whether use of the new plant variety in food or animal feed raises safety or regulatory issues with respect to the Federal Food, Drug, and Cosmetic Act (FD&C Act).
The Environmental Protection Agency (EPA) defines a plant-incorporated protectant (PIP) as “a pesticidal substance that is intended to be produced and used in a living plant, or the produce thereof, and the genetic material necessary for the production of such a pesticidal substance,” including “any inert ingredient contained in the plant, or produce thereof” (40 CFR 174.3). EPA regulates PIPs under the FD&C Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, the cry1Fv3 and cry1Ac(synpro) genes and resulting expression products in DAS-81419-2 soybean are considered pesticidal substances and the pat gene and its expression product are considered inert ingredients. Therefore, the safety assessment of these proteins falls under the regulatory purview of EPA.
Inheritance and Stability
Dow confirmed the genomic stability of the inserted DNA and its single-locus inheritance pattern. Genomic stability was demonstrated using Southern blot analysis of genomic DNA, which showed consistent hybridization patterns over five generations. Segregation ratios were determined using a lateral flow strip assay and event-specific PCR analysis targeting the inserted DNA. Dow reports that the results of Chi-square analysis of the segregation data support the conclusion that the inserted DNA is inherited according to Mendelian principles.
Food & Feed Use
Soybeans (Glycine max) are grown around the world for a variety of food, feed, and industrial uses. Soybean seeds are processed into meal and oil. Soybean meal is processed in moist heat to inactivate trypsin inhibitors and lectins, which are anti-nutrients occurring in raw soybeans. Most soybean meal is used in animal feed, primarily for poultry, swine, and beef and dairy cattle. A small fraction of soybean meal is processed into soy flours and soy proteins for a variety of human food uses. Soybean oil is rich in polyunsaturated fatty acids and is commonly used as a salad and cooking oil and in the production of margarine and other food ingredients. Whole soybeans are also used to prepare traditional foods such as tofu, miso, soymilk, tempeh, and soy sauce.
Scope of Analyses
Dow analyzed 88 components in forage and seed derived from DAS-81419-2 soybean and compares them to the variety (Maverick; control) used to develop DAS-81419-2 soybean. Dow also compares the composition of DAS-81419-2 soybean with six conventional varieties (reference varieties) grown under the same field conditions and with composition data obtained from publicly available sources.
Study Design - Compositional Analyses
Dow describes the study design used to obtain soybean forage and seed for compositional analysis. DAS-81419-2 soybean and the control were grown at ten locations in North America during the 2011 growing season. At each location, the varieties were planted in a randomized complete block design, with four replicate plots of each variety. Three of the six reference varieties were also grown at each field site such that each individual reference variety was grown at a total of five field sites. Dow states that appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop.
Forage samples (composite of the above-ground portion of three whole plants at the R3 growth stage) and seed samples (a representative 500 gram sample) were collected from each plot per site. Dow used analysis of variance methods to compare compositional data from DAS-81419-2 soybean forage and seed combined across all field sites, with data from the control. Statistical differences in composition were considered significant at the 95% confidence level (p < 0.05) after applying a statistical method intended to minimize the rate of false positives.1 When a statistically significant difference was detected between DAS-81419-2 soybean and the control, Dow assessed whether the difference was biologically meaningful with regard to food and feed safety. This assessment included a comparison of the values for DAS-81419-2 soybean with the range observed from the six reference varieties, as well as values in the published literature.2
Results of analyses
Compositional Analysis of Soybean Forage:
Dow reports forage compositional data for: crude protein, crude fat, ash, moisture, carbohydrates (by calculation), acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium, and phosphorus. No statistically significant differences were observed between DAS-81419-2 soybean and the control for these components. Further, the mean values for each of the DAS-81419-2 soybean components were within the combined range of the reference varieties and, when available, the published composition data for soybean.
Compositional Analysis of Soybean Seed:
Dow reports compositional data for 62 components in seed. Dow analyzed seed for proximates (crude protein, crude fat, ash, moisture, carbohydrates (by calculation), ADF, NDF, and total dietary fiber), minerals, amino acids, fatty acids, vitamins (Vitamins A, C, E, and B-vitamins), anti-nutrients (lectin, phytic acid, raffinose, stachyose, and trypsin inhibitor), and isoflavones (total daidzein, total genistein, and total glycitein).3 Statistically significant differences were observed in crude fat, ash, moisture, phenylalanine, palmitic acid, linolenic acid, eicosenoic acid, Vitamin B5, γ-tocopherol and total glycitein mean values when DAS-81419-2 soybean was compared with the control. However, where statistical differences were observed, the mean values for the components were within the combined range of the reference varieties and, when available, the published composition data for soybean.
Summary of Compositional Analyses
Dow concludes from its compositional analyses that forage and seed from DAS-81419-2 soybean are compositionally equivalent to forage and seed from conventional soybean varieties. Dow states that while statistically significant differences were observed between DAS-81419-2 soybean and the control, these differences were negligible relative to natural variation and the results were within ranges found for conventional soybean and are not biologically meaningful.
To assess levels of endogenous allergens in DAS-81419-2 soybean, Dow tested DAS-81419-2 soybean extracts for binding to allergen-reactive IgE by Western blot and by enzyme linked immunosorbent assay (ELISA) inhibition analysis. Dow obtained soy-reactive IgE from the pooled sera of ten clinically reactive patients. The developer observed no differences in endogenous allergen levels between DAS-81419-2 soybean and control seed extracts. Dow concludes that the genetic modification used to generate DAS-81419-2 soybean did not alter endogenous soybean allergenicity.
FDA evaluated Dow’s submission to determine whether DAS-41819-2 soybean raises any safety or regulatory issues with respect to its use in food or feed. Based on the information provided by the company and other information available to the agency, FDA did not identify any issues under the FD&C Act that would require further evaluation at this time.
Dow has concluded that DAS-81419-2 soybean and the foods and feeds derived from it are not materially different in composition, safety, or any other relevant parameter from soybean varieties now grown, marketed, and consumed in the United States. At this time, based on Dow’s data and information, the agency considers Dow’s consultation on DAS-41819-2 soybean to be complete.
Carrie McMahon, Ph.D.
1 Benjamini, Y., Hochberg, Y., (1995). Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society 57: 289-300.
2 Literature ranges were not reported for calcium and phosphorus in forage. In seed, literature ranges were not reported for total dietary fiber, selenium, Vitamin A, Vitamin B3, Vitamin B5, Vitamin B6, Vitamin C, and β-, γ-, δ-, and total tocopherol.
3 Dow states that 17 components were not statistically analyzed because greater than 50 percent of the samples were below the limit of quantitation. These components were sodium, caprylic acid, capric acid, lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, pentadecenoic acid, palmitoleic acid, heptadecanoic acid, heptadecenoic acid, γ-linolenic acid, eicosadienoic acid, eicosatrienoic acid, arachidonic acid, Vitamin A, and β-tocopherol.