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Biotechnology Consultation Note to the File BNF No. 000111

Return to inventory: Submissions on Bioengineered New Plant Varieties

See also Biotechnology: Genetically Engineered Plants for Food and Feed and about Submissions on Bioengineered New Plant Varieties


Date: September 9, 2008

Subject: Biotechnology Notification File (BNF) BNF 000111; GAT4621 and ZM-HRA proteins; Corn Transformation Event 98140; Unique Identifier DP-098140-6

Keywords: Corn, Zea mays (L.) , transformation event 98140, herbicide tolerance, glyphosate, glyphosate N-acetyltransferase, GAT, GAT4621 protein, gat4621 gene, Bacillus licheniformis, acetolactate synthase (ALS) (or acetohydroxyacid synthase or AHAS), ALS-inhibiting herbicides, ZM-HRA protein, zm-hra gene

Background

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.

Intended Effect

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.

Genetic Modifications and Characterization

Parental Variety

Pioneer used the proprietary inbred corn line, PHWVZ as the parental variety for transformation.

Transformation Plasmid and Transformation Method

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.

Table 1. Genetic elements contained in the T-DNA region of the plasmid PHP24279
Genetic element in T-DNADescription
Right BorderT-DNA Right Border region, from Ti plasmid of Agrobacterium tumefaciens
pinII TerminatorTerminator region from Solanum tuberosum proteinase inhibitor II gene
zm-hra GeneModified endogenous Zea mays acetolactate synthase gene
zm-als PromoterPromoter region from Zea mays acetolactate synthase gene
CaMV 35S EnhancerEnhancer region from the Cauliflower Mosaic Virus genome
ubiZM1 PromoterPromoter region from Zea mays ubiquitin gene
ubiZM1 5'UTR5' untranslated region from Zea mays ubiquitin gene
ubiZM1 IntronIntron region from Zea mays ubiquitin gene
gat4621 GeneSynthetic glyphosate N-acetyltransferase gene
pinII TerminatorTerminator region from Solanum tuberosum proteinase inhibitor II gene
Left BorderT-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.

Characterization, Inheritance, and Stability of the Introduced DNA

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.

Introduced Proteins

Identity and Function

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.

Expression Levels and Protein Characterization

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:

  • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm purity and molecular weight;
  • Western blot analysis to confirm equivalent molecular weight and immunoreactivity;
  • N-terminal amino acid sequence analysis to determine the identity of the proteins;
  • Mass determination of tryptic peptides by matrix assisted laser desorption ionization mass spectroscopy (MALDI-MS) to confirm identity of the proteins;
  • Glycoprotein staining to determine potential post-translational modification (i.e., glycosylation).

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.

Safety Assessment of the Introduced Proteins

GAT4621 Protein

To assess potential allergenicity of the GAT4621 protein, Pioneer used a weight of evidence approach.

  • Bioinformatics. When the amino acid sequence of the GAT4621 protein is compared to the amino acid sequences of known allergens in the FARRP6 database (University of Nebraska Allergen Database, Version 7, January 2007; www.allergenonline.com) using the FASTA34 sequence alignment program, the identified alignments did not exceed the 35% threshold within the 80 amino acid windows and there were no eight or greater contiguous identical amino acid matches between the GAT4621 protein and known allergens.
  • Simulated Gastric and Intestinal Fluid. When subjected to simulated gastric fluid (SGF), the GAT4621 protein is rapidly hydrolyzed to low molecular weight fragments in less than 30 seconds; and when subjected to simulated intestinal fluid (SIF), the GAT4621 protein, including the low molecular weight fragments seen in SGF, is completely and rapidly hydrolyzed in less than 5 minutes.
  • Glycosylation. Pioneer performed glycoprotein staining of plant and microbially derived GAT4621 protein which indicated that the GAT4621 protein is not glycosylated.
  • Potential Allergenicity of Donor Organism.
    • The DNA donor organism, B. licheniformis, has been a safe source of enzymes used in the food industry. FDA has affirmed the GRAS status of various enzyme preparations from B. licheniformis.
    • There have been reports of farmers' or mushroom workers' lung disease (extrinsic allergic alveolitis) caused by inhalation of spores from bacteria, including B. licheniformis. Pioneer states that this type of lung disease has been reported to be caused by inhalation of the bacterial spores themselves. Pioneer concludes that because the 98140 corn contains one specific protein derived from B. licheniformis there is no exposure to whole spores from the source organism in the 98140 corn and the reports of lung disease caused by Bacillus spores are not relevant to the allergenicity assessment for the GAT4621 protein.
    • There have been some reports of specific detergent enzymes (alpha-amylases and serine proteases) derived from Bacillus species, including B. licheniformis, producing occupational asthma in detergent industry workers. However, the GAT4621 protein is structurally and functionally distinct from enzymes derived from B. licheniformis reported to cause occupational asthma.

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.

ZM-HRA Protein

To assess potential allergenicity of the ZM-HRA protein, Pioneer used a weight of evidence approach.

  • Bioinformatics. When the amino acid sequence of the ZM-HRA protein is compared to the amino acid sequences of known allergens in the FARRP6 database (University of Nebraska Allergen Database, Version 7, January 2007; www.allergenonline.com) using the FASTA34 sequence alignment program, the identified alignments did not exceed the 35% threshold within the 80 amino acid windows and there were no eight or greater contiguous identical amino acid matches between the ZM-HRA protein and known allergens.
  • Simulated Gastric and Intestinal Fluid. When subjected to SGF, the ZM-HRA protein is rapidly hydrolyzed to low molecular weight fragments in less than 30 seconds; and when subjected to SIF, the ZM-HRA protein, including the low weight molecular fragments seen in SGF, is completely and rapidly hydrolyzed in less than 30 seconds.
  • Glycosylation. Pioneer performed glycoprotein staining of plant and microbially derived ZM-HRA protein which indicated that the ZM-HRA protein is not glycosylated.
  • Potential Allergenicity of the Donor Organism and Protein.
    The source of the zm-hra gene, which codes for the ZM-HRA protein, is corn. Pioneer states that corn has a long history of safe use for humans and animals and that the corn ALS protein, from which the ZM-HRA protein is derived, has not been characterized as an allergen. Pioneer further states that the ALS protein is also present in many species, including bacteria, fungi, algae, and higher plants, including crop plants.

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.

Food and Feed Use

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.

Compositional Analysis

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.

Table 2. Components measured in corn forage and grain
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).

Testing Strategy

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.

Forage Analysis

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.

Grain Analysis

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.

Proximates and Fiber

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.

Fatty Acids

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.

Amino Acids

Pioneer measured the levels of total amino acids, free amino acids, and five acetylated amino acids (NAA, NAG, NAThr, NASer, NAGly) in corn grain.

  • Total amino acids:
    No statistically significant differences between the 98140 corn and the non-transgenic control corn were observed in the mean levels of total amino acids in the grain, with the exception of the mean level of tryptophan. Based on the LMM approach, the mean level of tryptophan is statistically significantly higher in 98140 corn than in non-transgenic control corn. However, based on the FDR approach, this difference is not statistically significant. Pioneer noted that the mean levels of all amino acids were within the 99% tolerance interval and/or combined literature ranges.

     
  • Free amino acids:
    Pioneer states that amino acids in corn are divided between those that are incorporated into proteins and those in the free amino acid pool. The free amino acid pool in corn includes L-isomers of amino acids that are used in protein synthesis as well several amino acids that are not normally incorporated into proteins. The amino acids that play key roles in the incorporation into proteins and transfer of ammonia, such as glutamic acid and aspartic acid, are found in relatively high amounts, but the concentrations of the other free amino acids are very low. To clarify whether low levels of acetylation of amino acids affected the composition of the free amino acid pool in the 98140 corn, Pioneer measured individual free amino acids levels in the 98140 corn and the non-transgenic control corn. Two non-amino acid compounds (ethanolamine and ammonia) were measured along with the amino acids because they are recognized as analytes by the method used. There were no statistically significant differences in the mean levels of the free amino acids between the 98140 corn and the non-transgenic control corn. Furthermore, all mean levels were within the 99% tolerance intervals. Pioneer concluded that the free amino acid pools in the 98140 corn and the non-transgenic control corn were comparable.

     
  • Acetylated amino acids:
    According to a recent publication cited by Pioneer (Siehl et al, 2005), the GAT enzyme from B. licheniformis, similar to the GAT4621 enzyme, is able to acetylate seven amino acids (L-aspartate, L-glutamate, L-serine, phospho-L-serine, L-threonine, L-asparagine, and L-cysteine) with low catalytic efficiency. Pioneer states that the GAT4621 enzyme has low but measurable affinity for aspartate, glutamate, serine, threonine, and glycine. Because GAT4621 was expected to have the greatest effect on L-glutamate and L-aspartate, Pioneer initially measured the levels of NAA and NAG in corn grain and found that the mean levels of these amino acids were statistically significantly higher in the 98140 corn than in the non-transgenic control corn. The mean levels of NAA and NAG in the 98140 corn were 0.0403 % dw and 0.0079 % dw, respectively. For comparison, the mean levels of NAA and NAG in the non-transgenic control corn were 0.00009 % dw and 0.00005 % dw, respectively. Pioneer calculated that NAA and NAG make up less than 0.05 % dw of corn grain and less than 0.5% of the total amino acids in the 98140 corn grain.

    Pioneer subsequently measured the mean concentrations of NAThr, NASer, and NAGly in the 98140 corn grain. Although the levels of these acetylated amino acids were statistically significantly higher in the 98140 corn than in the non-transgenic control corn, they were more than 100 fold lower than the levels of NAA and NAG. For example, the mean levels of NAThr, NASer, and NAGly in the 98140 corn were less than 0.0003% on a dw basis. The overall level of all five acetylated amino acids in the 98140 corn grain was less than 0.05 % dw. Literature values for acetylated amino acids are not available. Pioneer states that NAThr, NASer, and NAGly are also present in non-transgenic control corn.

    Pioneer measured the levels of NAA and NAG in commonly consumed foods, some of which were selected on the basis of high concentrations of aspartic acid and glutamic acid.[3] NAA and NAG were found to be present in all tested foods, including fruits and vegetables, eggs, ground meat, milk, sardines, walnuts, chocolate, tea, roasted coffee beans, grains, and other foods. Pioneer concluded that NAA and NAG are normal components of the diet, based on their widespread presence in common foods.

    Pioneer also conducted an exposure assessment for humans to compare the estimates of dietary intake of NAA and NAG with and without exposure from the 98140 corn. Pioneer estimated that commercialization of the 98140 corn may increase the dietary exposure to NAA and NAG in the U.S. population above current levels of exposure.[4]

    Pioneer assessed the biological significance of increased levels of the acetylated amino acids in the human diet. Pioneer states that NAG, NAA, NAThr, NASer, and NAGly are naturally present in non-transgenic corn and that NAA and NAG are also present in a wide variety of commonly consumed foods. On the basis of the widespread distribution of NAA and NAG in foods, the overall low level of NAThr, NASer, and NAGly in the 98140 corn, Pioneer's estimate of dietary intakes of the 98140 corn, as well as the presence of deacetylases (aminoacylases that deacetylate N-acetylated amino acids) in the human body, Pioneer concluded that no safety issues are expected as a result of the estimated increased consumption of acetylated amino acids in the human diet due to commercialization of 98140 corn. Additionally, Pioneer cited its broiler study (described below) as further evidence of safety.

    Pioneer considered whether increases in dietary exposure to NAA and NAG resulting from commercialization of 98140 corn would have potential effects on individuals who may not express functional deacetylases, such as individuals with Canavan Disease (CD) or aminoacylase 1 deficiency. Individuals with CD do not express a functional aspartoacylase and are unable to deacetylate NAA. However, in CD the principle source of NAA is endogenous production in the brain. Pioneer states that the exposure to NAA from consumption of the 98140 corn is negligible compared to the amount of NAA produced in the brain and compared to the whole body pool of NAA in persons with CD. Consequently, Pioneer concluded that there will be no adverse impact to individuals with CD from the potential increase in dietary NAA as a result of consuming 98140 corn and that 98140 corn does not pose a safety concern for persons with CD. Individuals with aminoacylase 1 deficiency excrete elevated levels of N-acetylated amino acids in their urine. The elevated level of N-acetylated amino acids may be the result of incomplete metabolism of endogenous N-acetylated amino acids as well as N-acetylated amino acids from commonly consumed foods. The increase in exposure to acetylated amino acids due to consumption of the 98140 corn would be negligible compared to the amount of acetylated amino acids produced within the body and consumed normally in the diet. Pioneer concluded that 98140 corn is safe to consume for persons with aminoacylase 1 deficiency.

    Pioneer assessed the biological effects of increased levels of NAA and NAG in the broiler diet because broilers consume a significant portion of corn. Pioneer cites the results of its 42-day broiler study in which broilers were fed diets containing 98140 corn or non-transgenic control corn. Pioneer calculated exposure to NAA and NAG from diets containing the 98140 corn. The average dietary exposure to NAA was 21.7 mg/kg bw and to NAG was 8.0 mg/kg bw. No statistically significant differences were noted in mortality, weight gain, feed efficiency, and organ and carcass yield variables between broilers consuming diets produced with the 98140 corn and those consuming diets produced with the non-transgenic control corn, except for the kidney, as a percent of the live weight of the bird when conventional statistics were used. However, this parameter was not statistically significant when an adjusted p-value was used. All these variables also fell within the tolerance intervals derived from broilers fed diets containing the non-transgenic corn varieties. Pioneer concluded that no safety issues are expected as a result of the estimated increase in exposure to NAA and NAG in the broiler diet.

    Pioneer also considered the effect of the increased levels of NAA and NAG in all animal diets because about 52% of the corn grown in the United States is consumed by animals. The greatest percentage is consumed by beef cattle followed by poultry, swine, and dairy cows. Pioneer calculated that a 725 kg dairy cow eating corn grain would consume 5.0 and 1.0 mg/kg bw per day of NAA and NAG, respectively, and 8.2 and 1.2 mg/kg bw per day of NAA and NAG, respectively, when consuming silage. Pioneer similarly calculated that the predicted exposures for feeder steers and finisher steers eating both corn grain and corn silage would be 9.9 mg NAA and 1.7 NAG mg/kg bw per day, respectively. Pioneer concluded that the exposure levels of NAA and NAG for dairy and beef cattle diets containing the 98140 corn are lower than the levels broilers were exposed to in the 42-day broiler study. Additionally, Pioneer noted that microbes in the rumen of cattle are expected to metabolize NAA and NAG.

    Pioneer also considered the effect of the increased levels of NAA and NAG in swine diets where corn makes up approximately 44% of starter diets and 77% of grow-finish diets. Pioneer calculated that the level of exposure to NAA and NAG in starter swine would be 11.5 and 2.3 mg/kg bw, respectively; and, in growing/finishing swine, 12.8 and 2.5 mg/kg bw, respectively. Pioneer stated that these levels were much lower than those tested in the broiler study.

    Pioneer concluded that there are no safety issues that would be expected to result from the potential increase in human and animal exposure to these amino acids.

Minerals

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.

Vitamins

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

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.

Secondary Plant Metabolites

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.

Conclusions

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.