Biotechnology Consultation Note to the File BNF No. 000129
Return to inventory: Completed Consultations on Foods from Genetically Engineered Plant Varieties
Biotechnology Consultation - Note to the File
Biotechnology Notification File BNF No. 000129
April 27, 2012
DP-Ø73496-4, herbicide tolerant canola
Canola; Brassica napus; herbicide tolerance; DP-Ø73496-4 event; 73496 canola; gat4621 gene; glyphosate N-acetyltransferase (GAT) protein from Bacillus licheniformis; glyphosate; N-acetylglyphosate; Pioneer Hi-Bred International, Inc. (Pioneer); NPC 00005
This document summarizes our evaluation of Biotechnology Notification File (BNF) No. 000129. In a submission dated February 25, 2011, Pioneer Hi-Bred International, Inc. (Pioneer) submitted a safety and nutritional assessment of bioengineered herbicide tolerant canola, containing the transformation event DP-Ø73496-4 (hereafter referred to as 73496 canola). Pioneer provided additional information on September 20 and December 21, 2011, and on January 6 and April 6, 2012. FDA evaluated the information in Pioneer's submissions to ensure that regulatory and safety issues regarding human food and animal feed derived from the new plant variety have been resolved prior to commercial distribution.
In our evaluation of BNF No. 000129, we considered all information provided by Pioneer 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 technical effect of the modification in 73496 canola is to confer herbicide tolerance to glyphosate. To accomplish this objective, Pioneer introduced the gat4621 gene that encodes the glyphosate N-acetyltransferase (GAT4621) protein, which confers tolerance to glyphosate.(1) The GAT4621 protein renders the canola tolerant to glyphosate through acetylation of glyphosate to the non-phytotoxic N-acetylglyphosate.
The purpose of this evaluation is to assess whether Pioneer has introduced a substance into 73496 canola that would require premarket approval or raise other 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, the herbicide residues and metabolic by-products in 73496 canola, resulting from the detoxification of the applied herbicide by the expression product, are considered pesticidal residues. In its submission to FDA, Pioneer indicated that it submitted a tolerance petition and supporting residue data to EPA to amend the glyphosate tolerance to include N-acetylglyphosate for canola.
Genetic Modification and Characterization
Pioneer transformed the recipient canola line 1822B (a non-transgenic line developed by Pioneer) to obtain 73496 canola.
Introduced DNA and Transformation Method
Pioneer describes the PHP28181 plasmid, which contains one expression cassette within the PHP28181A fragment. The PHP28181A fragment was excised using restriction enzymes and isolated by gel purification. The gat4621 expression cassette contained the gat4621 gene under the control of the UBIQ10 promoter from Arabidopsis thaliana and the pinII terminator from Solanum tuberosum. The ampicillin resistance gene bla, present in the intact PHP28181 plasmid, is not expected to be present in the transformed cells because it is located outside of the isolated PHP28181A fragment.
To generate 73496 canola, Pioneer transformed canola microspores by particle bombardment with gold particles coated with the purified PHP28181A fragment. Following transformation, the microspores were cultured first under dark conditions and then under dim light. Green embryos were cultured at 4°Celsius (°C), then at 25°C in the presence of glyphosate to select for glyphosate-tolerant transformants. Transgenic plants (T0) were regenerated from the glyphosate-tolerant transformants.
Characterization, Inheritance, and Stability of the Introduced DNA
Pioneer characterized the DNA insert in 73496 canola using restriction enzyme digestion of genomic DNA followed by Southern blot analysis. Pioneer concludes that the results of this analysis demonstrate that 73496 canola contains a single intact copy of the gat4621 expression cassette in the canola genome. Pioneer also confirmed that no vector backbone sequences (i.e., sequences outside of the PHP28181A fragment) from the PHP28181 plasmid were detected in 73496 canola. Therefore, the bla gene was not inserted into the canola genome.
Pioneer studied the inheritance of the DNA insert by determining the gat4621 genotype and the glyphosate-tolerance phenotype in five generations of 73496 canola and analyzing the results using Chi square analysis. Pioneer reports that (1) all plants that were positive for the gat4621 gene were also tolerant to glyphosate and (2) the observed genotypic and phenotypic segregation ratios in three out of four segregating generations were consistent with the expected ratio. According to Pioneer, the results for one of the segregating generations were not consistent with the expected ratio; however, Pioneer explains that this generation was cross-pollinated by hand and it is likely that some flower buds may have self-pollinated. Based on the results of Chi square analysis, Pioneer concludes that the gat4621 genotype and the glyphosate-tolerance phenotype in 73496 canola segregate according to Mendel's laws of segregation for a single locus.
Pioneer assessed the stability of the DNA insert across five generations of 73496 canola using restriction enzyme digestion of genomic DNA followed by Southern blot analysis. Pioneer concludes, based on the results of this analysis, that a single copy of each of the cassette elements (UBQ10 promoter, gat4621 gene, and pinII terminator) was present and the integrity of the cassette was maintained across multiple generations.
Identity and Function of the Introduced Protein
Pioneer genetically engineered 73496 canola to express the glyphosate N-acetyltransferase (GAT4621) protein. The GAT4621 protein renders the plant tolerant to glyphosate through acetylation of glyphosate to the non-phytotoxic N-acetylglyphosate.
The GAT4621 protein is a variant of the GAT protein developed by Pioneer through DNA shuffling of three gat genes isolated from the Gram-positive soil bacterium Bacillus licheniformis. According to Pioneer, the GAT4621 protein is 75 – 78 percent identical and 90 – 91 percent similar at the amino acid level to each of the three native GAT proteins from which it was derived; however, the GAT4621 protein exhibits increased catalytic activity towards glyphosate relative to the native GAT proteins. The DNA sequence of the gat4621 gene used by Pioneer was also codon-optimized for expression in plants and is identical to the gat4621 coding sequence present in Pioneer's 98140 corn, the subject of BNF 000111. The plant-expressed GAT4621 protein is 147 amino acids in length, and has a molecular weight of 16.5 kDa.
Pioneer conducted expression studies of the GAT4621 protein in 73496 canola and a near isoline control variety.(2) Protein concentration was measured using enzyme-linked immunosorbent assay (ELISA) and reported for whole plant, root, and seed samples collected from plants from each of six locations in North America, with the exception that seed samples were only collected from five locations. Pioneer treated 73496 canola with two applications of glyphosate herbicide. Pioneer reports that the mean GAT4621 protein concentration ranged from 5 to 7 nanograms per milligram of tissue (ng/mg) dry weight in whole plant material across growth stages; the average concentration of the GAT4621 protein in root and seed samples fell within this range. The GAT4621 protein was not detected in the non-transgenic control tissues.(3)
Pioneer also measured GAT4621 protein concentration in processed 73496 canola meal (i.e., toasted meal fraction with hulls) and reported that the GAT4621 protein was present at a concentration of 2.5 ng/mg dry weight.
Safety Assessment of Potential for Toxicity and Allergenicity of the Introduced Protein
To obtain sufficient quantities of the GAT4621 protein for conducting safety assessment studies, Pioneer produced the GAT4621 protein in Escherichia coli. Pioneer confirmed the identity and biochemical equivalence of the GAT4621 proteins expressed in E. coli and 73496 canola, using several analytical techniques.(4) Based on the results of these studies, Pioneer concludes that E. coli-expressed GAT4621 protein is biochemically equivalent to the canola-expressed GAT4621 protein. E. coli-expressed GAT4621 protein was used subsequently as a proxy for the canola-expressed GAT4621 protein for in vitro digestion studies and an in vivo acute oral toxicity study.
To assess the potential for toxicity of the GAT4621 protein, Pioneer considered (1) the presence and safe use of the DNA donor B. licheniformis in food and in the production of food enzymes, (2) the similarity of the gat4621 amino acid sequence to the sequences of known toxins and (3) the results of an acute oral toxicity study in mice. Pioneer conducted bioinformatic analyses using the BLASTP 2.2.13 algorithm against Release 176.0 (February 15, 2010) of the National Center for Biotechnology Information Entrez Protein dataset. Pioneer identified similarities between the GAT4621 protein and other proteins in the dataset; however, none of the similar proteins identified were toxins. Pioneer reports the results of an acute oral toxicity study, in which a single dose of 1640 mg of GAT4612 protein per kilogram of body weight was administered to 10 mice, 5 per sex. Pioneer states that there was no evidence of toxicity during the 14 day observation period. Based on these results, Pioneer concludes that the GAT4621 protein is nontoxic to humans and other mammals.
To assess the potential for allergenicity of the GAT4621 protein, Pioneer considered the similarity of the gat4621 amino acid sequence to the sequences of known allergens, its glycosylation status, and its digestibility in simulated gastric and intestinal fluids. Pioneer used the Food Allergy Research and Resource Program Database (FARRP, version 10.0) to compare the amino acid sequences of the GAT4621 protein to known allergens. Pioneer reports that there were no matching alignments that met or exceeded 35 percent identity over 80-amino-acid stretches and no eight or greater contiguous identical amino acid matches observed with the GAT4621 protein. According to Pioneer, the GAT4621 protein is rapidly hydrolyzed in both simulated gastric fluid (less than 30 seconds) and simulated intestinal fluid (less than 5 minutes) and is not glycosylated in planta. Based on these results, Pioneer concludes that the GAT4621 protein is unlikely to be allergenic to humans and other animals.
Potential Substrates of gat4621 N-acetyltransferase Activity
Pioneer conducted a substrate specificity study of the GAT4621 protein for glyphosate as well as for various agrochemicals,(5) amino acids and antibiotics. The ability of the GAT4621 protein to N-acetylate the selected substrates was tested using assay conditions representative of physiological conditions in the plant cytosol and chloroplast. Pioneer reports that only five amino acid substrates had measurable enzyme activity: L-aspartate, L-glutamate, L-serine, glycine, and L-threonine. The level of catalytic efficiency of the GAT4621 protein for these amino acids was less than 1 percent of the activity on glyphosate.
Potential New Polypeptides
Pioneer assessed the potential for the creation of new open reading frames (ORFs) and for disruption of endogenous coding sequences resulting from the insertion of the DNA fragment. Pioneer screened the sequence spanning the insertion and genome junction for the presence of new ORFs and identified one putative new ORF of 14 amino acids in length. The new ORF was evaluated using bioinformatic methods (i.e., amino acid sequence comparison) for its similarity to known toxins, allergens, antinutrients, or pharmacologically active proteins; no matches were found. Other data presented in the submission show that insertion of the gat4621 expression cassette did not generate novel proteins or alter expression of existing genes in a way that impacted safety. These data included results from Southern blot analysis showing the intact integration of the gat4621 expression cassette; Western blot analysis confirming expression of the GAT4621 protein as expected without the expression of unexpected proteins of different sizes; and compositional analyses of key components demonstrating that 73496 canola was comparable to commercial canola lines.
Food & Feed Use
Pioneer states that 73496 canola will be grown for the same uses as currently commercialized canola.
Canola (Brassica napus or B. campestris (B. rapa)) refers to low-erucic acid, low-glucosinolate rapeseed varieties. Canola seeds are processed into oil and meal, which are used primarily for food and feed, respectively; industrial uses of canola are limited. Canola oil is low in saturated fatty acids and is commonly used as cooking oil for frying and baking, among other food applications. Canola meal is the fraction of the whole canola seed remaining after the seed is crushed and its oil extracted. The majority of canola meal is used in animal feed: primarily for cattle, dairy cows, and pigs and, to a lesser extent, for poultry, aquaculture, lamb and other livestock. In addition, the use of protein isolates from canola meal in potential human food applications(6) has been described in published literature. The typical processing steps for production of canola oil and meal include cooking, flake pressing, solvent extraction, and toasting.
Scope of Analyses
Pioneer analyzed the composition of seed from 73496 canola; a non-transgenic, near isoline variety (hereafter referred to as the control); and non-transgenic canola lines that are currently or were previously used for commercial canola oil production (hereafter referred to as the reference varieties).(7)
Study Design - Compositional Analyses
In a study conducted in 2009, Pioneer analyzed seed from 73496 canola and the control grown at five locations in North America, with four replicates at each location, using a randomized complete block design. According to Pioneer, 73496 canola plants were treated with glyphosate in a manner consistent with typical agronomic practices. In a separate study conducted in 2008 and 2009, Pioneer analyzed seed from the five reference varieties grown at five locations to establish statistical tolerance intervals for each component. The composition data in both studies included proximates (moisture, crude protein, crude fat, ash, and carbohydrates (by difference)), fiber (acid detergent fiber (ADF), neutral detergent fiber (NDF), and crude fiber), fatty acids, amino acids, vitamins, minerals, glucosinolates, secondary metabolites, phytosterols, and anti-nutrients. Pioneer also provides information on acetylated amino acids in seed, whole plant, and processed meal and oil fractions.
Pioneer performed statistical analyses on composition data obtained from 73496 canola and control samples using mixed models for across site analysis. Pioneer reports the composition data analyses by providing mean values, ranges, p-values, and p-values adjusted using a False Discovery Rate (FDR) procedure.(8) A significance level of p < 0.05 was chosen for both analyses. Pioneer calculated a statistical tolerance interval using the compositional results from the reference varieties in order to establish the normal variation for components in canola grown commercially in North America.(9)
Results of analyses - Compositional analyses of canola seed
Proximates and Fiber
Pioneer reports the compositional analyses for proximates and fiber. No statistically significant differences (adjusted p-value) were observed between 73496 canola and the control. Furthermore, the mean concentrations for the analyzed proximates and fiber were within the tolerance interval established using the reference varieties.
Pioneer reports the compositional analyses for sixteen fatty acids. Of these, statistically significant differences (adjusted p-value) between 73496 canola and the control were observed for oleic and linoleic acids. However, the mean levels for all the analyzed fatty acids, including oleic and linoleic acids, were within the tolerance interval established using the reference varieties.
Total and Free Amino Acids
Pioneer reports compositional analyses for eighteen total amino acids and summarizes the results of compositional analyses for several free amino acids. Based on the results obtained, Pioneer concludes that there were no statistically significant differences (adjusted p-value) between 73496 canola and the control for the total amino acids but that there were statistically significant differences for some of the free amino acids. However, the mean concentrations for all the amino acids analyzed were within the tolerance interval established using the reference varieties.
Acetylated Amino Acids
As a result of the findings of the GAT4621 protein substrate specificity study, Pioneer also reports the compositional analyses for five acetylated amino acids in 73496 canola and the control: N-acetylated aspartate (NAA), N-acetylated glutamate NAG), N-acetylated serine, N-acetylated glycine, and N-acetylated threonine. Results were reported for samples from seeds, whole plants, and processed fractions, including toasted meal (with and without hulls) and refined, bleached and deodorized (RBD) oil (with and without hulls). Acetylated amino acids were not detectable or were below the limit of quantification in the RBD oil. Statistically significant differences were reported for several acetylated amino acids between 73496 canola and the control, with NAA and NAG showing increased concentrations in seeds, whole plants, and toasted meal fractions. In toasted 73496 canola meal with hulls (the typical processed canola product consumed by animals), the mean concentrations of NAA and NAG were 2872 mg per kilogram (mg/kg) and 62 mg/kg, respectively. For comparison, in toasted control canola meal with hulls, the mean concentrations of NAA and NAG were 2.31 mg/kg and 1.36 mg/kg, respectively.
Pioneer considered the safety of human and animal exposure to acetylated amino acids in general and to NAA and NAG in particular. Pioneer describes acetylated amino acids as normal components of food and feed that have histories of safe use. Using a hypothetical total replacement scenario (assuming100 percent of the canola meal in human and animal diets were derived from 73496 canola), Pioneer estimated the dietary exposure of humans and livestock to acetylated amino acids from consumption of toasted 73496 canola meal with hulls. Pioneer estimated a human daily intake of 0.44 mg NAA/kg body weight per day (mg/kg bw/d) and 0.01 mg NAG/kg bw/d;(10) Pioneer estimated the mean broiler chicken daily intake to be 39.1 mg NAA/kg bw/d and 0.85 mg NAG/kg bw/d. Pioneer compared these estimated dietary intakes to the "no observed adverse effect level" for NAA of 451.6 mg/kg bw/d and for NAG of 914 mg/kg bw/d, established in rat oral toxicity studies. Pioneer concludes that there are no safety issues that would be expected to result from animal dietary exposure to 73496 canola meal or human dietary exposure to protein isolates derived from 73496 canola meal.(11)
Vitamins and Tocopherol
Pioneer reports the compositional analyses for six B vitamins, four isoforms of tocopherol, and total tocopherol. Of these, statistically significant differences (adjusted p-value) between 73496 canola and the control were observed for delta-tocopherol and total tocopherol. However, the mean concentrations for the measurable B vitamins, the individual tocopherol isoforms, and total tocopherol were within the tolerance intervals established using the reference varieties.(12)
Pioneer reports the compositional analyses for nine minerals. Of these, a statistically significant difference (adjusted p-value) between 73496 canola and the control was observed for magnesium. However, the mean concentrations for the analyzed minerals, including magnesium, were within the tolerance intervals established using the reference varieties.
Pioneer reports the compositional analyses for thirteen individual glucosinolates as well as for total glucosinolates. Of these, a statistically significant difference (adjusted p-value) between 73496 canola and the control was observed for progoitrin. However, the mean concentrations for the measurable glucosinolates, including progoitrin and total glucosinolates, were within the tolerance interval established using the reference varieties.(13)
Antinutrients and Phytosterols
Pioneer reports the compositional analyses for tannins (soluble and insoluble), sinapine, phytic acid, several phytosterols, and total sterols. Of these, a statistically significant difference (adjusted p-value) between 73496 canola and the control was observed for cholesterol. However, the mean concentrations for the analyzed anti-nutrients and phytosterols, including cholesterol and total sterols, were within the tolerance interval established using the reference varieties.
In summary, the magnitude of differences between 73496 canola and the control were small and within the respective tolerance intervals established using the reference varieties, with the exception of acetylated amino acids. Although levels of NAA and NAG in 73496 canola were increased compared to the control and reference varieties, Pioneer concludes that the estimated dietary exposure would not result in safety concerns. Based on the results of the compositional analyses, Pioneer concludes that 73496 canola is otherwise compositionally equivalent to the control and to canola varieties that are currently or were previously grown for commercial use.
FDA evaluated Pioneer's submission to determine whether 73496 canola raises any safety or regulatory issues with respect to the intended modification or with respect to the food and feed itself. Based on the information provided by Pioneer 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.
Pioneer concludes that its herbicide tolerant canola variety, 73496 canola, and the food and feed derived from it are as safe as conventional canola varieties, and with the exception of the herbicide tolerance trait, are not materially different in composition or other relevant parameters from other canola varieties now grown, marketed and consumed in the United States. At this time, based on Pioneer’s data and information, the agency considers Pioneer’s consultation on 73496 canola to be complete.
Carrie McMahon, Ph.D.
(1)Pioneer submitted its evaluation of the potential for allergenicity and toxicity of the GAT4621 protein, which FDA designated as New Protein Consultation No. NPC 00005 under FDA's Guidance to Industry: "Recommendations for the Early Food Safety Evaluation of New Non-Pesticidal Proteins Produced by New Plant Varieties Intended for Food Use." FDA responded that it had no questions regarding Pioneer's conclusions that GAT4621 protein is neither toxic nor allergenic.
(2)For the GAT4621 protein expression studies, Pioneer used 73496 canola plants from the F1 generation of 5536F x 1822R (bearing event 73496) canola and a control variety derived from 5536F x 1822R canola.
(3)Pioneer reports that the lower limits of quantification (LLOQ) by ELISA were 0.29 ng/mg dry weight for whole plant samples and 0.22 ng/mg dry weight for root and seed samples.
(4)The analytical techniques discussed in the submission include sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western hybridization analysis, N-terminal amino acid sequence analysis, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).
(5)Pioneer explains that it uses the term "agrochemicals" to refer to compounds that have fungicidal, herbicidal, or insecticidal activity and that are currently applied to row crops.
(6)To date, FDA has evaluated and responded to two GRAS notices on the use of canola protein isolates (GRAS Notice No. GRN 000327 and GRN 000386). FDA had no questions about the notifiers' conclusions that the intended uses of canola protein isolates in human food are GRAS.
(7)For the compositional analyses, Pioneer used 73496 canola plants from the F1 generation of 5536F x 1822R (bearing event 73496) canola and control plants derived from 5536F x 1822R canola.
(8)According to Pioneer, the number of false positive outcomes increases as the number of analytes increases. Pioneer states that, in order to help manage the false positive rate, the FDR method was applied to account for making multiple comparisons.
(9)Pioneer states that the statistical tolerance interval was calculated to contain 95 percent of the values in the population of commercial canola, with 95 percent confidence.
(10)Pioneer used disappearance data from the World Health Organization Global Environment Monitoring System to estimate human dietary intake of 73496 canola meal.
(11)FDA has considered the safety of acetylated amino acids as components of food and feed previously. See biotechnology consultations BNF 000108 and BNF 000111.
(12)Pioneer reports that the values for beta-tocopherol were below the LLOQ for 73496 canola and the control and that a tolerance interval could not be calculated from the reference varieties due to insufficient values being detected above the LLOQ.
(13)Pioneer reports that the values for four of the glucosinolates were below the LLOQ for 73496 canola and the control and that a tolerance interval could not be calculated from the reference varieties for three of these glucosinolates due to either no values or an insufficient number of values being detected above the LLOQ.