Novel Food Information – Glyphosate tolerant Corn – HCEM485

Health Canada has notified Stine Seed Farm, Inc. that it has no objection to the food use of corn expressing a version of corn 5-enolpyruvylshikimate-3-phosphate synthase to confer tolerance to glyphosate herbicide. The Department conducted a comprehensive assessment of this corn event according to its Guidelines for the Safety Assessment of Novel Foods. These guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

The following provides a summary of the notification from Stine Seed Farm, Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

HCEM485 corn was genetically engineered using aerosol beam direct DNA transfer to insert four copies of a 6 kbp fragment derived from maize B73 inbred line containing the EPSPS coding sequence. This fragment was identified from a genomic library cloned into a bacterial artificial chromosome (BAC). The epsps gene was modified by site-directed mutagenesis resulting in the introduction of two amino acid changes, threonine to isoleucine at amino acid position 102 and proline to serine at amino acid position 106. This mutated version of EPSPS, termed 2mEPSPS, is 99.7 % similar at the amino acid level to previously approved genetically modified plants, namely maize GA21 (Monsanto, 1999) and cotton GHB614(Bayer, 2008). The sequences were provided by the petitioner and are available in the public domain.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada’s Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents (e.g. Codex Alimentarius). The assessment considered: how HCEM485 corn was developed; how the composition and nutritional quality of HCEM485 corn compared to non-modified varieties; and what the potential is for HCEM485 corn to be toxic or cause allergic reactions. Stine Seed Farm has provided data that demonstrates HCEM485 corn is as safe and of the same nutritional quality as traditional corn varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from HCEM485 corn are considered novel foods under the following part of the definition of novel foods:

• "c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that
  • (i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism".

2. Development of the modified plant

The epsps gene was isolated from a bacterial artificial chromosome (BAC) library of genome sequence fragments derived from the maize inbred line B73. The clone containing epsps was identified by screening with a DNA probe complementary to part of the epsps gene. The cassette contained 5’ transcriptional and translational control elements; the epsps coding sequence, comprised of eight exons and seven introns and including a predicted chloroplast transit peptide; and a 3’ untranslated region also for expression regulation. Using conventional molecular techniques, the expression cassette was cloned into a plasmid vector and subject to site-directed mutagenesis to introduce the same two amino acid substitutions introduced into the 2mEPSPS in GA21 corn. The double mutated 2mEPSPS also contains a serine at position 89 to match the sequence of native corn EPSPS.

Following mutagenesis, the plasmid was amplified and digested, and the fragment containing the 2mEPSPS expression cassette was separated from the plasmid backbone by purification from agarose gel. The DNA fragment was introduced into corn embryo tissue by aerosol beam injection, a method for delivery of naked DNA, i.e. without reliance on a biological vector or other carrier. The target cells derived from an elite inbred corn line, Stine 963. Following insertion, the cells were recovered in non-selective medium and then subjected to successive rounds of growth in selective medium containing increasing levels of glyphosate.

3. Characterization of the Modified Plant

In order to estimate the number of 2mEPSPS expression cassette insertions, characterize the insert locus, and confirm the absence of plasmid backbone, the genome of HCEM485 corn was analysed by Southern blot. The data provided were supportive of a proposed model of four complete tandem copies of the insert at a single locus, where the third copy is oriented in the reverse direction relative to the others. From the Southern blot analysis, no incomplete fragments of the expression cassette were detected. The plasmid backbone was not detected in HCEM485. Due to the repetitive nature of the insert region of HCEM485, the petitioner was unable to provide a more complete molecular characterization of the insert locus such as nucleotide sequence data.

A trait segregation experiment was conducted to provide evidence for the number of insert loci in HCEM485. The initial HCEM485 line (designated T0) was self fertilized for two successive generations (T1 and T2). The T2 generation homozygous progeny were crossed with corn line 9289 to yield a hybrid generation (F1) of plants hemizygous for the insert. The F1 progeny were then crossed with corn line 9032 to yield a second generation, which was again self-fertilized to produce an F2 generation. All progeny plants were subject to screening for the herbicide resistance trait by application of 2.5 times the recommended level of glyphosate for field use. The proportion of trait positive to trait negative plants for each generation was compared to the expected ratios for presence of the gene copies at a single locus. Agreement between expected and observed values was calculated using a chi-squared test with Yates’ correction factor. Overall, the data presented support the petitioner’s claim that the glyphosate tolerance trait encoded by 2mEPSPS agrees with a model for single locus inheritance, indicating that the expression cassette was inserted at one location in the HCEM485 genome.

4. Product Information

The host and donor plants are of the same species, Zea mays L., which has a millennia-long history of use as food, and is cultivated around the world. The inserted DNA includes the 2mEPSPS coding sequences and maize-derived expression regulation sequences; no other heterologous, non-maize sequences were inserted. Additionally, no vector organism (e.g. Agrobacterium) was used to mediate the DNA insertion. The possibility of transgenic sequences being present in HCEM485 was therefore discounted. No safety concerns are noted given the history of safe food use of the host and donor organism.

The 2mEPSPS expressed in HCEM485 is of similar amino acid sequence to EPSPS expressed endogenously by various plants and microbes. The petitioner cited a 2009 study which reported that a public database search returned more than 5000 EPSPS proteins expressed in plants and microorganisms. A 2013 review^{Footnote 1}  by Hammond and colleagues was also cited, which stated that to date, no EPSPS proteins have been reported to exhibit toxic effects.

The mechanism of action of glyphosate (N-(phosphonomethyl) glycine) involves the inhibition of the shikimate pathway, which functions in the biosynthesis of aromatic amino acids that are required for plant survival. Specifically, glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme that catalyzes the transfer of a phosphate group from phosphoenolpyruvate to 3-phosphoshikimate to produce 5-enolpyruvylshikimate-3-phosphate (EPSP). The herbicide tolerance trait is conferred by incorporating a mutated version of EPSPS (2mEPSPS) that is not inhibited by glyphosate.

No characterization of 2mEPSPS protein expression in plant tissues was provided. The petitioner rationalized that the level of expression in target tissues was likely to not be informative given that the expression regulation sequences were the same as native EPSPS.

5. Dietary Exposure

The genetic modification of HCEM485 corn is not intended to alter corn consumption patterns. Therefore, the use of herbicide tolerant HCEM485 corn and products derived from it will be similar to traditional corn varieties. The introduction of HCEM485 corn will only replace a fraction of current corn varieties and is not anticipated to result in a change in the dietary intake of corn-derived products.

6. Nutrition

The petitioner provided compositional data to assess key nutritional parameters in forage and grain samples from HCEM485 maize line and control hybrids. Maize line HCEM485 and three conventional control hybrids were grown in 2007 in the United States at locations representative of the range of environmental conditions under which maize is typically grown (i.e. the United States Corn Belt). The control hybrids were produced by crossing inbred lines, each previously used in the breeding of line HCEM485. All plants were grown according to local agronomic practices in a randomized block design, with three replicates for each genotype per location. HCEM485 hybrids were treated with glyphosate herbicide at the 3-4 leaf stage (Roundup WeatherMAX) at the usual commercial application rate. Samples of maize grain from each of the individual control hybrids were pooled for analysis for each replicated plot. After processing, the samples were analysed according to methods published and approved by AOAC, or other industry standard analytical methods. Data for analytes above the limit of quantification were subjected to analysis of variance across all locations with genotype and location as factors. For each variable, the statistical significance of the genotype effect (i.e. HCEM485 vs. control hybrids) was determined using a standard F-test.

The petitioner analyzed the grain samples for 87 parameters including all proximates, amino acids, fatty acids, micronutrients, naturally occurring anti-nutritional factors, etc. mentioned in the OECD’s “Consensus Document on Compositional Consideration for New Varieties of Maize (Zea mays).” Of the 65 analyte comparisons that were suitable for statistical analysis, 45 showed no statistically significance differences. The highest mean differences (>10%) between maize line HCEM485 and the control hybrids were observed for copper (HCEM485 was 13.01% lower than controls), vitamin B1 (HCEM485 was 12.23% lower than controls), folic acid (HCEM485 was 15.25% lower than controls), alpha-tocopherol (HCEM485 was 13.38% lower than controls), delta-tocopherol (HCEM485 was 18.19% lower than controls), gamma-tocopherol (HCEM485 was 12.46% lower than controls), total tocopherols (HCEM485 was 12.75% lower than controls), and inositol (HCEM485 was 13.36% lower than controls).

The petitioner concluded that where statistically significant differences were observed, the magnitudes of the differences were considered small, and all nutrients analyzed were found to be within the range of natural variation reported in the literature (ILSI crop database and OECD). Additionally, the petitioner concluded that no consistent patterns emerged to suggest that biologically significant changes in composition of the grain or forage had occurred as an unintended consequence of the genetic modification.

It was found that the quantity and quality of the information provided was satisfactory. The differences between maize line HCEM485 and the control hybrids noted in the compositional data do not raise any nutritional or safety concerns.

7. Chemistry/Toxicology

No data for chemical contaminants, including data for heavy metals, persistent organic pollutants (POPs) or mycotoxins in the plants were included in the petition. Given the understanding of the mode of action for this trait, the introduced genetic change is not expected to alter the susceptibility of HCEM485 plants to mycotoxin producing fungi, or to uptake chemical contaminants compared to conventional corn varieties. The presence of HCEM485 in the Canadian food supply would not be of unique health significance from a chemical safety perspective relative to conventional corn.

The petitioner did not submit independent toxicological studies for 2mEPSPS. Instead, an argument was presented that the 2mEPSPS expressed in HCEM485 was equivalent to that in other events for which the results of toxicology studies are available in the public domain.

The case for equivalence rests on the data demonstrating the identity of the protein, its biochemical structure, and its function or activity. The identity of 2mEPSPS is established by the deduced amino acid sequence. 2mEPSPS expressed in HCEM485 is identical to that expressed in GHB614 and GA21 except for a single amino acid substitution that renders the HCEM485 version more similar to the native maize EPSPS. The level of identity between these proteins is 99.7 % at the amino acid level. An E. coli-expressed His(6)-tagged 2mEPSPS used in Western blots was of the same sequence as GHB614 and GA21 2mEPSPS. The Western blot analyses supplied evidence of structural equivalency and conservation between 2mEPSPS between E. coli-expressed His(6)-tagged 2mEPSPS, and 2mEPSPS expressed in GA21 and in HCEM485. Using the monoclonal antibody, it was possible to distinguish 2mEPSPS from native EPSPS common across many bacteria and plant species. The detection of 2mEPSPS in test samples as well as EPSPS in non-transformed corn using polyclonal antibodies suggested that a constellation of epitopes is preserved and that overall structure is maintained across both mutant and native forms of EPSPS. Functional equivalence was established by expression of the glyphosate herbicide tolerance trait. Taken together, the evidence supports the claim for equivalency between the 2mEPSPS expressed in HCEM485 and GHB614 and GA21 events, as well as the E. coli-expressed His(6)-tagged 2mEPSPS.

The petitioner submitted published summaries of toxicity studies conducted with GA21 corn and GHB614 cotton. These included a publicly available safety assessment by ANZFA (now FSANZ; 2000)^{Footnote 2} , which assessed much of the same information reviewed by Health Canada, and a more recent review article by Hérouet-Guicheney et al. (2009)^{Footnote 3} .

The review article by Hérouet-Guicheney et al. (2009) supports the safety of 2mEPSPS demonstrated in earlier studies. It was reported that there were no adverse effects in mice administered a dose of 2000 mg/kg bw of 2mEPSPS protein that had been produced in E. coli. Equivalence was established between the bacterially-produced protein with that in cotton event GHB614, as well as transgenic canola and soybean events. The protein characterization methods used included sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC) – electron spray spectroscopy (LC/MS), N-terminal sequencing, and Western blotting. The authors also conducted digestibility studies on the bacterially produced 2mEPSPS protein and conducted database searches for homology with known toxins and allergens.

Based on the equivalency data and the information provided, no concerns were identified with respect to the toxicological characterization of modified EPSPS enzyme expressed in corn line HCEM485.

8. Allergenicity

The AllergenOnline database was also queried, and 2mEPSPS was not found to be similar to any known allergens using an 80-mer sliding window for 8 amino acid epitopes. The bioinformatics analysis results support the claim that 2mEPSPS is not allergenic.

9. Conclusion

Health Canada’s review of the information presented in support of the food use of HCEM485 corn did not raise concerns related to food safety. Health Canada is of the opinion that food derived from HCEM485 corn is as safe and nutritious as food from current commercial corn varieties.

Health Canada’s opinion deals only with the food use of HCEM485 corn. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the potential environmental and animal health issues associated with HCEM485 corn. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective with the commercial sale of products derived from HCEM485 corn.