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In a submission dated June 28, 2007, Pioneer Hi-Bred International, Inc. (Pioneer), a DuPont company, submitted to FDA a safety and nutritional assessment of genetically engineered dual herbicide tolerant corn, transformation event 98140 (hereafter referred to as 98140 corn). Pioneer submitted additional information in submissions dated September 18, 2007; November 29, 2007; February 14, 2008; March 12, 2008; and July 10, 2008.[1] Pioneer concluded that food and feed derived from 98140 corn are as safe and nutritious as food and feed derived from conventional corn.
The intended effect of the modification is to confer tolerance to both glyphosate and acetolactate synthase (ALS)-inhibiting herbicides. To accomplish this objective, Pioneer introduced a glyphosate N-acetyltransferase (gat4621) gene, derived from the sequences of three gat genes from Bacillus licheniformis (B. licheniformis), and a modified acetolactate synthase (zm-hra) gene, derived from the corn als gene, into the Pioneer proprietary inbred corn line, PHWVZ. The GAT4621 protein, encoded by the gat4621 gene, confers tolerance to glyphosate-containing herbicides. The ZM-HRA protein, encoded by the zm-hra gene, confers tolerance to the ALS-inhibiting class of herbicides.
Pioneer used the proprietary inbred corn line, PHWVZ as the parental variety for transformation.
Pioneer constructed plasmid PHP24279 to contain two expression cassettes; the gat4621 gene that encodes the GAT4621 protein, and the zm-hra gene that encodes the ZM-HRA protein. Pioneer produced the 98140 corn via Agrobacterium tumefaciens-mediated transformation of PHWVZ using plasmid PHP24279. The functional genetic elements contained within the T-DNA regions of plasmid PHP24279 are provided in Table 1.
| Genetic element in T-DNA | Description |
|---|---|
| Right Border | T-DNA Right Border region, from Ti plasmid of Agrobacterium tumefaciens |
| pinII Terminator | Terminator region from Solanum tuberosum proteinase inhibitor II gene |
| zm-hra Gene | Modified endogenous Zea mays acetolactate synthase gene |
| zm-als Promoter | Promoter region from Zea mays acetolactate synthase gene |
| CaMV 35S Enhancer | Enhancer region from the Cauliflower Mosaic Virus genome |
| ubiZM1 Promoter | Promoter region from Zea mays ubiquitin gene |
| ubiZM1 5'UTR | 5' untranslated region from Zea mays ubiquitin gene |
| ubiZM1 Intron | Intron region from Zea mays ubiquitin gene |
| gat4621 Gene | Synthetic glyphosate N-acetyltransferase gene |
| pinII Terminator | Terminator region from Solanum tuberosum proteinase inhibitor II gene |
| Left Border | T-DNA Left Border region, from Ti plasmid of Agrobacterium tumefaciens |
Transformed embryos were subsequently grown in cell culture using medium containing glyphosate for selection of cells expressing the gat4621 transgene. After two weeks, calli that demonstrated resistance to glyphosate were identified and polymerase chain reaction (PCR) analysis was performed to verify the presence of the inserted gat4621 and zm-hra genes. The embryonic calli were regenerated into whole transgenic plants and evaluated for glyphosate and ALS-inhibitor herbicide tolerance.
Pioneer conducted Southern blot analysis to characterize the DNA insertion in the 98140 corn. Pioneer states that the analysis confirmed that a single, intact copy of DNA containing the gat4621 and zm-hra expression cassettes has been inserted into the corn genome. Pioneer's analysis verified that the integrity of the inserted DNA containing the gat4621 and zm-hra cassettes was maintained upon integration. Southern blot analysis also demonstrated the absence of plasmid backbone DNA from outside the T-DNA region.
Pioneer used Chi-square analysis of trait inheritance data from four generations (BC0S1, BC1S1, BC2 and BC3) to determine the heritability and stability of the gat4621 and zm-hra genes in the 98140 corn. In order to confirm the expected segregation ratios, Pioneer performed PCR analysis on leaf punches from seedlings. Pioneer states that the results of this analysis are consistent with the finding of a single locus of insertion of the gat4621 and zm-hra genes that segregates in the 98140 corn progeny according to Mendel's laws of genetics.
Pioneer also conducted Southern blot analysis on three additional generations, BC0S2, BC1, and BC1S1, to verify that the insert in the 98140 corn remained intact and stably integrated across generations. Pioneer states that results confirmed the stability of the insertion in the 98140 corn across four breeding generations.
Pioneer noted that the 98140 corn was genetically engineered to express two proteins, the glyphosate N-acetyltransferase GAT4621 protein and the acetolactate synthase ZM-HRA protein, that render the transgenic plant tolerant to two classes of herbicides.
The GAT4621 protein is encoded by the gat4621 gene, which was developed by DNA shuffling of three gat genes isolated from the common soil bacterium B. licheniformis, a Gram positive saprophytic bacterium that is widespread in nature. The protein is 75-78% identical and 90-91% similar at the amino acid level to each of the three native GAT proteins from which it was derived. The GAT4621 protein is 147 amino acids in length and has an approximate molecular weight of 17 kilodaltons (kDa). There are 32 to 36 amino acid changes (22 or 23 of which are conservative) in the shuffled GAT4621 protein, depending on which of the three original native B. licheniformis GAT proteins is used for comparison. The GAT4621 protein acetylates (and inactivates) glyphosate more efficiently than the native B. licheniformis enzymes.
The ZM-HRA protein is a modified version of the native corn acetolactate synthase protein. The ZM-HRA protein confers tolerance to the ALS-inhibiting class of herbicides. ALS-inhibiting herbicides function by inhibiting branched-chain amino acid biosynthesis. The herbicide tolerant zm-hra gene was made by isolating the endogenous corn als gene and introducing two specific amino acid changes (P165A and W542L). The locations of these two substitutions are equivalent to the locations of commonly found natural tolerance mutations reported in the scientific literature. The two mutations together result in a higher level of tolerance to ALS inhibiting herbicides compared to single amino acid changes. The full length ZM-HRA protein, which includes a chloroplast transit peptide, is 638 amino acids and has an approximate molecular weight of 69 kDa. The mature protein has 598 amino acids and a predicted molecular weight of 65 kDa.
Pioneer measured and reported the levels of the GAT4621 and ZM-HRA proteins in leaf, root, pollen, forage, grain, and whole plant tissue samples collected from plants grown at six field locations in North America. The samples were collected at various growth stages with relevance to commercial corn production practices. Three replicated samples per tissue per location were collected for the 98140 corn and one sample per tissue per location was collected for near isogenic, non-transgenic control corn.[2] The samples were analyzed using enzyme linked immunosorbent assay (ELISA). Pioneer reports that the mean levels of GAT4621 protein in various plant tissues at various growth stages ranged from 2.6 (root) to 51 (leaf) nanograms per milligram (ng/mg) of tissue dry weight (dw). The mean levels of ZM-HRA protein in various tissues ranged from below the level of detection (pollen) to 6.70 (leaf) ng/mg of tissue dw. Neither the GAT4621 nor the ZM-HRA proteins were detected in non-transgenic control corn tissues sampled from the six locations.
In order to obtain sufficient quantity of the GAT4621 and ZM-HRA proteins for conducting safety assessment studies of each protein, Pioneer produced GAT4621 and ZM-HRA in, and purified from, an Escherichia coli (E. coli) protein expression system. The ZM-HRA protein was produced in its mature form without the chloroplast transit peptide sequence. Pioneer used immunoaffinity chromatography to purify small amounts of GAT4621 and ZM-HRA proteins from 98140 corn leaf tissue. Pioneer characterized the microbially expressed GAT4621 and ZM-HRA proteins, and demonstrated that the plant-derived GAT4621 and ZM-HRA proteins are equivalent to their microbially expressed counterparts.
To confirm the identity and equivalency of the E. coli-produced and corn-produced GAT4621 and ZM-HRA proteins, Pioneer used the following methods:
Based on the results of these studies, Pioneer concluded that the E. coli-produced and corn-produced GAT4621 and ZM-HRA proteins were equivalent. The E. coli-produced GAT4621 and ZM-HRA proteins were subsequently used for in vitro and in vivo biochemical and toxicological studies.
To assess potential allergenicity of the GAT4621 protein, Pioneer used a weight of evidence approach.
Pioneer concluded that the GAT4621 protein is not likely to cause an allergic reaction.
To assess potential toxicity of the GAT4621 protein, Pioneer conducted an amino acid homology search of the GAT4621 amino acid sequence against the sequences of known protein toxins in the National Center for Biotechnology Information (NCBI) protein database containing all entries from GenBank nucleotide translations and protein sequences from SWISS-PROT, PIR, PRF, and PDB databases. Pioneer concluded that the GAT4621 protein did not share relevant sequence similarities with known protein toxins, and was therefore unlikely to be a toxin itself.
Pioneer also conducted an acute oral toxicity study in mice. A single dose of 1640 milligrams per kilogram of body weight (mg/kg bw) of E. coli-produced and purified GAT4621 protein was administered by oral gavage to five male and five female mice. No clinical symptoms of toxicity, body weight loss, gross organ lesions, or mortality were observed. Pioneer concluded that the results of this study show that the GAT4621 protein is not acutely toxic.
Pioneer also noted that the GAT4621 protein retains the characteristics found in other N-acetyltransferases that are ubiquitous in plants and microorganisms. Although GAT4621 is an optimized protein, it is 75-78% identical and 90-91% similar at the amino acid level to the translated protein sequences of each of the three original B. licheniformis gat alleles from which the GAT4621 protein was derived.
Pioneer concluded that the GAT4621 protein is not toxic.
To assess potential allergenicity of the ZM-HRA protein, Pioneer used a weight of evidence approach.
Pioneer concluded that the ZM-HRA protein is not likely to cause an allergic reaction.
To assess potential toxicity, Pioneer conducted an amino acid homology search of the ZM-HRA amino acid sequence, using the BLASTP algorithm, against the sequences in the NCBI protein database containing all entries from GenBank nucleotide translations and protein sequences from SWISS-PROT, PIR, PRF, and PDB databases. Pioneer concluded that the ZM-HRA protein did not share relevant sequence similarities with known protein toxins and was therefore unlikely to be a toxin itself.
Pioneer also conducted an acute oral toxicity study in mice. A single dose of 1236 mg/kg bw of E. coli-produced and purified ZM-HRA protein was administered by oral gavage to five male and five female mice. No clinical symptoms of toxicity, body weight loss, gross organ lesions, or mortality were observed. Pioneer concluded that the results of this study show that the ZM-HRA protein is not acutely toxic.
Pioneer concluded that the ZM-HRA protein is not toxic.
Corn grain and its processed fractions are consumed as human food and animal feed. The majority of corn is used as animal feed, and a small percentage is harvested as forage and made into silage and fed to ruminants. The remainder of corn is exported, processed into food products, or converted to ethanol. Corn can be processed by wet and dry milling processes to convert the grain into food, feed, and fuel products.
Pioneer analyzed the composition of forage and grain from the transgenic 98140 corn and a near isogenic, non-transgenic control corn to assess whether the composition of the transgenic corn differs from that of non-transgenic control corn. Pioneer analyzed corn forage for proximates (protein, fat, and ash), amino acids, free amino acids, acetylated amino acids (N-acetylaspartate (NAA), N-acetylglutamate (NAG)), acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium and phosphorus. Pioneer analyzed corn grain for proximates, ADF, NDF, fatty acids, total amino acids, acetylated amino acids (NAA and NAG, N-acetylthreonine (NAThr), N-acetylserine (NASer), N-acetylglycine (NAGly)), free amino acids, minerals, vitamins, antinutrients, and secondary plant metabolites. The components measured in forage (see asterisked analytes) and grain are listed in Table 2.
| Proximates* & Fiber | Fatty Acids* | Total Amino Acids* and Acetylated Amino Acids* | Free Amino Acids* and Other Amino Compounds* | Minerals | Vitamins | Anti-Nutrients | Secondary Plant Metabolites |
|---|---|---|---|---|---|---|---|
|
protein fat ash moisture ADF NDF |
palmitic (16:0) palmitoleic (16:1) heptadecanoic (17:0) stearic (18:0) oleic (18:1) linoleic (18:2) linolenic (18:3) arachidic (20:0) eicosenoic (20:1) behenic (22:0) lignoceric (24:0) |
methionine cystine lysine tryptophan threonine isoleucine histidine valine leucine arginine phenylalanine glycine alanine aspartic acid glutamic acid proline serine tyrosine NAA NAG NAThr NASer NAGly |
L-aspartic acid L-threonine L-serine L-asparagine L-glutamic acid L-glutamine L-cysteine L-proline glycine L-alanine L-valine L-cystine L-methionine L-isoleucine leucine L-tyrosine L-phenylalanine γ-amino-n-butyric acid L-ornithine L-tryptophan L-lysine L-histidine L-arginine ethanolamine ammonia |
calcium copper iron magnesium phosphorus potassium sodium zinc |
beta-carotene vitamin B1 (thiamin) vitamin B2 (riboflavin) vitamin B3 (niacin) vitamin B6 (pyridoxine) folic acid α-tocopherol |
raffinose phytic acid trypsin inhibitor |
furfural p-coumaric acid ferulic acid |
| *The levels of the following fatty acids were below the limit of quantitation: caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), myristoleic (14:1), pentadecanoic (15:0), pentadecenoic (15:0), heptadecenoic (17:1), heptadecadienoic (17:2), γ-linolenic (18:3), nonadecanoic (19:0), eicosadienoic (20:2), eicosatrienoic (20:3), arachidonic (20:4), heneicosanoic (21:0) erucic (22:1), and tricosanoic acid (23:0). | |||||||
The forage and grain samples were collected from plants grown in 2006 at six field locations in corn-growing areas of North America. In a separate experiment, forage and seed tissue were collected from four different conventional commercial corn hybrids (reference corn) grown in 2003 at six field locations. In both experiments, the corn plants were grown using a randomized complete block design of two-row plots with three and four replicates at each location for 2006 and 2003, respectively.
Pioneer used compositional data derived from the reference corn to calculate tolerance intervals that contain 99% of the values obtained for each component with 95% confidence. Pioneer states that the compositional analysis of the reference corn helps to establish the normal variation in the levels of the measured components.
Pioneer performed the statistical analysis on compositional data obtained for the 98140 corn and the non-transgenic control corn. Pioneer used a conventional linear mixed model (LMM) approach to account for the design effects of location and blocks within location. In order to manage the false discovery rate, Pioneer employed the false discovery rate (FDR) approach (Benjamini and Hohberg, 1995; Westfall et al., 1999). A statistically significant difference between the mean level of each component in the 98140 corn and non-transgenic control corn was established at the FDR-adjusted p-value < 0.05. For each measured component, Pioneer provided the mean level (from all locations), range, FDR adjusted p-value, p-value, tolerance interval and the combined range of values for each analyte from the published literature (combined literature range), including on-line databases. Pioneer then compared the compositional data for the 98140 corn to the non-transgenic control, the 99% tolerance level, and the combined literature ranges.
Pioneer assessed the composition of corn forage harvested at the R4 maturity stage by measuring proximates (protein, fat, and ash), minerals, carbohydrates, ADF and NDF, amino acids, free amino acids, and the acetylated amino acids NAA and NAG. No statistically significant differences between the 98140 corn and the non-transgenic control corn were observed in the mean levels of proximates, minerals, carbohydrates, ADF, NDF, total and free amino acids and mean levels were also within the 99% tolerance intervals and combined literature ranges. Literature values and statistical tolerance intervals were not available for the amino acid content of corn forage. However, FDA notes that most of the means for the specific amino acids fell within or close to the literature ranges for corn silage published by OECD (2002).
As expected, the mean levels for NAA and NAG were statistically significantly higher (p < 0.05) for the 98140 corn compared to the non-transgenic control corn. No literature data were found regarding the level of the two acetylated amino acids in corn forage. In order to address the biological significance of the elevated levels of the two acetylated amino acids, Pioneer investigated whether the amount of the acetylated amino acids would significantly affect the free amino acid pool. The free amino acids in corn forage were measured for both the non-transgenic control corn and the 98140 corn and no significant differences were detected in the mean levels of any of the free amino acids between the non-transgenic control corn and the 98140 corn. Pioneer states that NAG and NAA make up less than 1.2 % of the total amino acids in the 98140 corn forage and that the protein levels and free amino acid pool are comparable to the non-transgenic control corn. Pioneer states that the low levels of acetylation of aspartate and glutamate in the forage of the 98140 corn are not affecting amino acid incorporation into proteins or the level of the free amino acid pool.
Pioneer concluded that forage from 98140 corn is comparable to that from non-transgenic control corn.
Pioneer assessed the composition of corn grain harvested at the R6 maturity stage by measuring components listed in Table 2. The results reported by Pioneer are summarized below.
Pioneer measured the levels of proximates, ADF and NDF in corn grain. No statistically significant differences between 98140 corn and the non-transgenic control corn were observed in the mean levels of protein, ash, NDF and ADF. All mean levels were within the 99% tolerance intervals and the combined literature ranges.
Pioneer measured the levels of fatty acids in corn grain. No statistically significant differences between the 98140 corn and the non-transgenic control corn were observed in the mean levels of any of the fatty acids. The means for all fatty acids measured in the 98140 corn and the non-transgenic control corn were within the 99 % tolerance interval and the combined literature range.
Pioneer measured the levels of total amino acids, free amino acids, and five acetylated amino acids (NAA, NAG, NAThr, NASer, NAGly) in corn grain.
Pioneer analyzed corn grain for the commonly occurring minerals. Pioneer found no statistically significant differences in the mean levels for the 98140 corn and non-transgenic control corn. In addition, the mean levels of all minerals for the 98140 corn and non-transgenic control corn were within the 99% tolerance intervals and combined literature ranges.
Pioneer analyzed corn grain for the vitamins listed in Table 2. Pioneer found no statistically significant differences between the mean level of these vitamins in the 98140 corn and the non-transgenic control corn. Moreover, all mean values for the 98140 corn and non-transgenic control corn were within the 99% tolerance intervals[5] and combined literature ranges.
Antinutrients that occur in corn grain include the non-digestible carbohydrate raffinose, phytic acid, and trypsin inhibitor. Pioneer reported that the mean levels of these antinutrients were not significantly different in the 98140 corn compared to the non-transgenic control corn and were within the 99% tolerance intervals and combined literature ranges.
Pioneer measured the secondary metabolites furfural,[6] p-coumaric acid, and ferulic acid in 98140 corn and the non-transgenic control corn. Pioneer states that the mean levels of these secondary metabolites were not statistically significantly different between the 98140 corn and the non-transgenic control corn.
Pioneer concluded that the 98140 corn is comparable in composition to non-transgenic control corn.
Pioneer has concluded that its dual herbicide tolerant corn variety, 98140 corn, and the foods and feeds derived from it are not materially different in safety, composition, or any other relevant parameter from corn varieties now grown, marketed, and consumed. At this time, based on Pioneer's data and information, the agency considers Pioneer's consultation on 98140 corn to be complete.
Karin Ricker, Ph.D.
[1] The GAT4621 protein was the subject of New Protein Consultation (NPC) 0005, and the ZM-HRA protein was the subject of NPC 0006, both of which were submitted by Pioneer. Pioneer incorporates the information submitted in NPC 0005 and NPC 0006 by reference in BNF 000111.
[2] The non-transgenic control corn has a genetic background similar to that of the 98140 corn but does not contain the gat4621 and zm-hra genes.
[3] Pioneer selected high aspartic acid and glutamic acid foods using the United States Department of Agriculture Nutrient Database for Standard Reference (Release 19; 2006).
[4] Pioneer used the Dietary Exposure Evaluation Model (DEEM)/Food Commodity Intake Database (FCID), Version 2.14, Exponent Inc., Washington, D.C. to estimate human exposure to NAA and NAG. Pioneer estimated exposure to NAA and NAG for the United States population as well as several populations subgroups, including children of 1-6 years of age. For the 98140 corn, Pioneer postulated two scenarios in which they assumed that 40 and 100 percent, respectively, of the commodity corn grown in the U.S. would be the 98140 corn.
[5] Tolerance intervals were not available for vitamin B2, vitamin B3, vitamin B6.
[6] Tolerance intervals were not available for furfural.
