This document summarizes our evaluation of biotechnology notification file (BNF) No. 000139. In a submission dated August 28, 2012, Syngenta Seeds, Inc. (Syngenta) and Bayer CropScience AG (BCS) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of their genetically engineered herbicide-tolerant soybean, transformation event SYHT0H2 (hereafter referred to as SYHT0H2 soybean). Syngenta and BCS (the developers) provided additional information on February 25, 2013. FDA evaluated the information in the developers’ submissions to ensure that regulatory and safety issues regarding the food or feed derived from the new plant variety have been resolved prior to commercial distribution.
In our evaluation, we considered all information provided by the developers 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 effects of the modification in SYHT0H2 soybean are to confer tolerance to p-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides and to glufosinate-ammonium. Syngenta and BCS introduced three genes: avhppd-03 derived from Avena sativa (common oat) and two variants of pat from the soil bacterium Streptomyces viridochromogenes. The avhppd-03 gene encodes AvHPPD-03 and the pat genes encode phosphinothricin N-acetyltransferase (PAT).
The purposes of this evaluation are (1) to assess whether the developers have introduced a substance requiring premarket approval as a food additive into food or feed and (2) to determine whether use of the new plant variety in food or feed raises other regulatory issues under the Federal Food, Drug, and Cosmetic Act (FD&C Act).
The Environmental Protection Agency (EPA) regulates herbicides under the FD&C Act and the Federal Insecticide, Fungicide, and Rodenticide Act. Under EPA regulations, herbicide residues and any metabolic by-products of herbicides present in herbicide-treated SYHT0H2 soybean are considered pesticide residues. In their submission to FDA, Syngenta and BCS refer to already established tolerances for use of glufosinate-ammonium in or on soybean commodities and for pre-emergence use of mesotrione (an HPPD-inhibiting herbicide) in soybean. The developers state that they will submit a regulatory package to EPA for post-emergence use of mesotrione on SYHT0H2 soybean.
The genetic elements intended for introduction (i.e., T-DNA sequences between the left and right border regions of pSYN15954) include three expression cassettes:
Following infection of immature soybean seed explants, Syngenta and BCS regenerated plantlets under conditions designed to eliminate A. tumefaciens and to select for transformants carrying the pat gene. Successful transformants were identified using real-time PCR analysis targeting the introduced genetic elements (avhppd-03 and pat genes).
Syngenta and BCS characterized the insert in SYHT0H2 soybean using nucleotide sequencing and Southern blot analysis of restriction enzyme digested genomic DNA. The results of these analyses demonstrate that the introduced DNA is located at a single insertion site and consists of two inverted fragments of the pSYN15954 T-DNA. The 5′ fragment lacks the entire avhppd03 cassette, left and right borders, and part of the pat-03-01 35S promoter. The 3′ fragment is largely intact, lacking the left and right borders and some upstream avhppd-03 regulatory elements. The SYHT0H2 soybean genome contains, in total, one copy of the avhppd-03 gene, two copies of the pat-03-01 gene, and two copies of the pat-03-02 gene.
Based on Southern blot analysis using probes complementary to the plasmid backbone, Syngenta and BCS conclude that SYHT0H2 soybean lacks plasmid backbone sequences. The developers conclude that the spectinomycin resistance gene from pSYN15954 is not present in the SYHT0H2 soybean genome.
Syngenta and BCS determined the genomic stability and inheritance pattern of the inserted DNA. Genomic stability was demonstrated using Southern blot analysis of genomic DNA, which showed consistent hybridization patterns over three generations. Segregation ratios were determined using real-time PCR analysis targeting the inserted DNA; segregation data was analyzed using Chi-square analysis. The results of the analyses of three SYHT0H2 soybean generations showed that the introduced DNA is inherited according to Mendelian principles.
To determine the site of genome insertion, the developers first sequenced the genomic regions flanking the insert in SYHT0H2 soybean and then used the results to sequence the corresponding region in the untransformed variety “Jack.” They searched publicly available databases for known coding sequences similar to the flanking sequences. Syngenta and BCS conclude that the insertion does not disrupt known soybean genes.
Identity and Function of Introduced Proteins
SYHT0H2 soybean was genetically engineered to express AvHPPD-03, which binds to HPPD-inhibiting herbicides with lower affinity than native soybean HPPD, thereby conferring tolerance to HPPD-inhibiting herbicides such as mesotrione. HPPD is in the tyrosine catabolic pathway, and it occurs in plants, animals, and bacteria. In plants, the reaction product of HPPD is homogenistate; homogenistate is the aromatic precursor of tocopherol and plastoquinone, which are essential to photosynthetic systems. Syngenta and BCS state that the amino acid sequence of AvHPPD-03 is 99.7% identical to that of HPPD in common oat.
SYHT0H2 soybean was also engineered to express PAT, which confers tolerance to the herbicide glufosinate-ammonium. PAT acetylates L-phosphinothricin (the active form of glufosinate-ammonium), thereby inactivating the herbicide. According to the developers, the two pat genes (pat-03-01 and pat-03-02) encode identical proteins, although their nucleic acid sequences differ slightly.
Protein Expression Levels
Syngenta and BCS conducted field studies to measure expression levels of AvHPPD-03 and PAT in SYHT0H2 soybean. Tissue samples were collected from SYHT0H2 soybean and control soybean grown at four locations in Argentina during the 2011-2012 growing season. The developers measured levels of the introduced proteins by enzyme linked immunosorbent assay (ELISA) in leaves, roots, forage, and seed samples collected at multiple growth stages. Analysis showed that concentrations of both proteins vary widely. For example, AvHPPD-03 concentration in seed (dry weight) ranged from 0.62-28.30 micrograms per gram (µg/g) tissue with a mean of 7.16 µg/g. PAT concentration in seed (dry weight) ranged from 0.07-14.85 µg/g with a mean of 2.70 µg/g. In general, the concentrations of both proteins were higher in vegetative-stage leaves and lower in roots, forage, and seeds.
Safety Assessment of the Introduced Proteins
Potential for Toxicity and Allergenicity of AvHPPD-03:
Syngenta and BCS use a weight-of-evidence approach to assess the safety of AvHPPD-03. The safety assessment includes discussions on the source of the protein, the protein’s function, and its potential to be a toxin or allergen.
HPPD proteins are present in common food crops such as soybean, maize, and oat, but they differ in their sensitivity to HPPD-inhibiting herbicides. AvHPPD-03 is derived from common oat, a food crop that is not known to have toxins or allergens. Given its similarity (99.7%) to HPPD in common oat, the AvHPPD-03 present in SYHT0H2 soybean is not expected to be allergenic or toxic.
Syngenta and BCS compared the amino acid sequence of AvHPPD-03 with the sequences of known toxins and allergens in the National Center for Biotechnology Information, Entrez Protein Database (NCBI, 2012) and in the Food Allergy Research and Resource Program, Allergen Protein Database (FARRP, v. 12.0). Based on these analyses, AvHPPD-03 is similar to HPPD orthologs in many plants and to other closely related proteins (i.e. glycoxylases and dioxygenase superfamily members). Some similarity was noted to bacterial HPPDs that are annotated as putative hemolysins. The metabolic product of HPPD is homogentisic acid, which under certain conditions may oxidize, polymerize, and be converted to compounds with hemolytic properties. Since AvHPPD-03 is more similar to plant HPPDs already present in food than it is to bacterial HPPDs, Syngenta and BCS conclude that the similarity to bacterial HPPDs is not evidence of toxicity.
The developers report the results of an acute oral toxicity study in mice and of in vitro
studies of protein stability. The results of the toxicity study show that AvHPPD-03 is not acutely toxic based on the absence of observable adverse effects at a level of 2000 milligrams per kilogram bodyweight, the highest level tested.2
Likewise, the protein’s rapid digestion in simulated gastric and intestinal fluids, and its loss of enzymatic activity and immunoreactivity at 65 degrees Celsius and above, supports the developers’ conclusion that AvHPPD-03 is not likely to be an allergen.
Based on the available evidence, Syngenta and BCS conclude that AvHPPD-03 is not likely to pose a health risk through consumption of SYHT0H2 soybean.
Potential for Toxicity and Allergenicity of PAT:
Syngenta and BCS use a weight-of-evidence approach to assess the safety of PAT, which the developers note is identical to the S. viridochromogenes-derived PAT protein introduced into several food crops that have a history of safe use. In their safety assessment, the developers discuss the source of the protein, the protein’s function, and evidence related to its potential to be a toxin or allergen. Syngenta and BCS conclude that the evidence shows that PAT is unlikely to be toxic or allergenic and that it does not pose a health risk through consumption of SYHT0H2 soybean.
Potential New Proteins
Syngenta and BCS analyzed the DNA sequences of the insert and adjoining genomic DNA to identify novel putative open reading frames (ORFs) generated by the insertion event. The analysis predicted 47 putative new ORFs, each defined by a known or putative start codon, stop codon, and coding sequence of at least 30 amino acids. According to the developers, 46 of the 47 putative ORFs were not positioned near a promoter, ruling out expression of all but one of the ORFs. The remaining ORF showed no relevant similarity to known allergens or toxins in the appropriate databases.
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 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 further processed into soy flours and soy proteins for a variety of 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
Syngenta and BCS analyzed 73 components in forage and seed derived from SYHT0H2 soybean and compare them to the variety (Jack; the control) used to develop SYHT0H2 soybean. Syngenta and BCS also compare the composition of SYHT0H2 soybean with composition data from six conventional, commercial varieties (reference varieties) grown under the same field conditions and with composition data obtained from publicly available sources.3
Study Design - Compositional Analyses
SYHT0H2 soybean was grown alongside the control variety and six reference varieties at eight locations in North America during the 2010 growing season, in areas where commercial soybeans are typically grown. The six reference varieties were included to establish a range of normal values for the components analyzed. At each location, the varieties were planted in a randomized complete block design, with four replicate plots of each variety. According to the developers, the plots were managed according to local agricultural practices.
Forage samples (composites of the above-ground portion of 10 plants at the R6 growth stage) and seed samples (composites of seeds from 30 plants at the R8 growth stage) were collected at each site. The developers state that the components analyzed were consistent with those recommended by the Organisation for Economic Co-Operation and Development (OECD).4
Syngenta and BCS used analysis of variance methods to compare composition values from SYHT0H2 soybean with values from the control. Statistical differences in composition were considered significant at
p < 0.05. When a statistically significant difference was detected between SYHT0H2 soybean and control, Syngenta and BCS assessed whether the difference was biologically meaningful with regard to food and feed safety and nutrition. This assessment included a comparison of the values for SYHT0H2 soybean with the range observed for the six reference varieties, as well as with values published in the ILSI Crop Composition Database.5
Results of analyses
Compositional Analysis of Soybean Forage:
Syngenta and BCS analyzed the compositional data for seven components in soybean forage (moisture, crude protein, crude fat, ash, carbohydrates (by calculation), acid detergent fiber (ADF), and neutral detergent fiber (NDF)). No statistically significant differences in forage composition were observed between SYHT0H2 soybean and the control. Further, the mean values for both varieties were within the combined range of the reference varieties.
Compositional Analysis of Soybean Seed:
Syngenta and BCS analyzed compositional data for 73 components in seed including: proximates (moisture, crude protein, crude fat, ash, and carbohydrates (by calculation)), fiber (ADF and NDF), minerals (Ca, Fe, Mg, P and K), vitamins (Vitamins A, B1, B2, B9, andK1), tocopherols
(α-, β-, γ-, and δ-tocopherols), tocotrienols, 18 amino acids, and 22 fatty acids. In addition, the developers analyzed seed for anti-nutrients (lectin, phytic acid, raffinose, stachyose, and trypsin inhibitor) and isoflavones (daidzein, glycitein, and genistein). They report the results of statistical analyses for 60 components in soybean seed.
Statistically significant differences were found between SYHT0H2 soybean and the control in ADF, iron, potassium, α-tocopherol, γ-tocopherol, δ-tocopherol, and in a majority of the amino acids and fatty acids. In all cases except for α-tocopherol, γ-tocopherol, and δ-tocopherol, the magnitudes of the differences between the mean values for SYHT0H2 soybean and the control were less than 10%. Syngenta and BCS also note that the mean levels for these components in SYHT0H2 soybean were within the combined range of reference varieties.
Syngenta and BCS report that although the mean value for α-tocopherol were lower by 11.6%, and the mean values for γ- and δ-tocopherols were higher by 12.4% and 29.1% in SYHT0H2 soybean compared with the mean values for the control, they fell within the range of values for the reference varieties and the ILSI database.8
Syngenta and BCS also discuss that the increased levels of γ- and δ-tocopherol levels in SYHT0H2 soybean seed compared with the control are consistent with similar reports in the published literature regarding the overexpression of HPPD in plants.
Summary of Compositional Analyses
Based on their compositional and nutritional analyses, Syngenta and BCS conclude that forage and seed from SYHT0H2 soybean are not materially different from those of conventional soybean varieties. The developers state that while the data from seed indicate that SYHT0H2 soybean differs slightly in composition from the control, the observed differences in composition were generally small and within the combined range of component levels for the six reference varieties.
Poultry Feeding Study
Syngenta and BCS provide summary data from a 42-day broiler chicken feeding study comparing poultry diets that contained soybean meal (29.0% to 33.5% of the total diet) derived from: (1) SYHT0H2 soybean, (2) a near-isogenic control, or (3) a commercial soybean variety. Based on the individual nutrient analyses of each of the processed meals, Syngenta and BCS adjusted the diets to the age-related nutritional needs of the chickens. The developers state that chickens fed diets prepared with SYHT0H2 soybean meal did not exhibit any adverse or unexpected effects and had similar performance parameters when compared to chickens fed the control- or commercial soybean variety-based diets.
Syngenta and BCS used mass spectrometry to measure the concentration of 12 known soybean allergens in SYHT0H2 soybean and compared them to levels in the control and to a tolerance interval calculated from data obtained from 17 reference varieties. In addition, the developers compared antibody binding to soybean proteins extracts from SYHT0H2 soybean, Jack, and two other reference varieties using allergen-reactive IgE from five soybean-reactive patients. Based on the results of these studies, Syngenta and BCS conclude that SYHT0H2 soybean is comparable to conventional soybean varieties with respect to seed content of known allergens and that SYHT0H2 soybean will not pose different health risks to soybean-allergic consumers than conventional soybean.
FDA evaluated Syngenta and BCS’s submission to determine whether SYHT0H2 soybean raises any safety or regulatory issues with respect to the intended modification or with respect to its use in food and feed. Based on the information provided by the companies and other information available to the agency, FDA did not identify any safety or regulatory issues under the FD&C Act that would require further evaluation at this time.
Syngenta and BCS have concluded that SYHT0H2 soybean and the foods and feeds derived from it are not materially different in composition, safety, or any other relevant parameter from other soybean varieties now grown, marketed, and consumed in the United States. At this time, based on Syngenta and BCS’s data and information, the agency considers the consultation on SYHT0H2 soybean to be complete.
Carrie McMahon, Ph.D.
Syngenta and BCS explain that the synthetic minimal plant promoter includes the TATA box and an adenine-rich sequence involved in transcription initiation from the Cestrum yellow leaf curling virus promoter, linked to a sequence from the region that is 3′ to the TATA box of the 35S promoter.
The acute oral toxicity study of AvHPPD-03 protein in mice was conducted using a microbially-produced AvHPPD-03 protein. Syngenta and BCS report that the SYHT0H2 soybean- and microbially-produced AvHPPD-03 proteins differ by a 4 amino acid, N-terminal truncation; however, they are otherwise biochemically and functionally equivalent, as based on molecular weight, immunoreactivity, and enzymatic activity.
Literature ranges were not reported for ADF and NDF (in forage) and for Vitamin A, Vitamin K1, β-tocopherol, γ-tocopherol, δ-tocopherol, and the tocotrienol isoforms (in seed).
OECD Consensus Document on Compositional Considerations for New Varieties of Soybean: Key Food and Feed Nutrients and Anti-Nutrients (2001).
Published in version 4.2 of the International Life Sciences Institute (ILSI) Crop Composition Database, accessed July 14, 2011, http://www.cropcomposition.org.
The developers state that the values for 13 of the 22 fatty acids were below the limit of quantitation in all replicate samples of SYHT0H2 soybean.
The developers state that some or all values for Vitamin A, b-tocopherol, and the four tocotrienol isoforms were below the limit of quantitation. Hence the means could not be calculated or statistically compared.
The developers note that tocopherol levels in soybean vary widely due to genetic and environmental influences.