Double Herbicide Tolerant Soybean Event FG72 (MST-FGØ72-2)

Health Canada has notified Bayer CropScience Inc. that it has no objection to the food use of double herbicide tolerant soybean event FG72. The Department conducted a comprehensive assessment of this variety 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.

Background:

The following provides a summary of the notification from Bayer CropScience Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Bayer CropScience Inc. has developed a herbicide tolerant soybean (Glycine max L.) event that is tolerant to the herbicide glyphosate and to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides. This soybean event was developed through biolistic-mediated transformation of soybean calli with a 7.3-kb DNA fragment (i.e., SalI fragment). This fragment contains the coding sequences (i.e., 2mepsps and hppdPfW336) and required regulatory elements to express the 2mEPSPS and HPPD W336 proteins, respectively. The 2mepsps coding sequence was generated by introducing mutations into the native epsps gene from maize (Zea mays L.), leading to a double mutant EPSPS protein with two amino acid substitutions (i.e., 2mEPSPS). This modification confers glyphosate tolerance by decreasing the binding affinity of the 2mEPSPS protein for glyphosate, allowing the protein to maintain sufficient enzymatic activity in the presence of the herbicide. The native hppd gene was isolated from Pseudomonas fluorescens, a Gram-negative rod-shaped bacterium that is ubiquitous in nature and often found in water, soil, and the plant rhizosphere. The hppdPfW336 coding sequence differs from the native Pseudomonas gene by a single amino acid substitution (i.e., glycine substituted to tryptophan).

The 2mEPSPS protein has been previously assessed by Health Canada and the Canadian Food Inspection Agency (CFIA) in Bayer CropScience Inc.'s GlyTol™ cotton event GHB614. EPSPS functions as part of the shikimate pathway used by bacteria, fungi, and plants for the biosynthesis of aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan).

According to the petitioner, HPPD proteins are ubiquitous in nature across all kingdoms: bacteria, fungi, plants, and animals (including mammals). In bacteria and mammals, HPPD proteins are involved in the catabolic degradation of tyrosine. Tyrosine is first converted to 4-hydroxyphenylpyruvate within the organism. HPPD enzymatic activity leads to the formation of homogentisate, which in plants is a precursor of plastoquinones and tocopherols essential to the photosynthetic transport chain and antioxidative systems. Inhibition of HPPD proteins results in destabilization of photosynthesis, subsequent bleaching of the foliage, and death of the plant. The W336 mutation increases HPPD tolerance to HPPD inhibitor herbicides by altering the binding site for these herbicides, thus decreasing binding affinity.

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 in this area (e.g., Codex Alimentarius). The assessment considered: how double herbicide tolerant soybean event FG72 was developed; how the composition and nutritional quality of this soybean event compared to conventional varieties; and the potential for this soybean event to be toxic or cause allergic reactions. Bayer CropScience Inc. has provided data that demonstrates that double herbicide tolerant soybean event FG72 is as safe and of the same nutritional quality as conventional soybean 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 the Food and Drug Regulations (Division 28). Double herbicide tolerant soybean event FG72 is considered a novel food 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

1. 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 petitioner has provided information describing the methods used to develop double herbicide tolerant soybean event FG72, in addition to the molecular biology data that characterize the genetic change, which results in the improved tolerance to glyphosate and HPPD inhibitor herbicides. This phenotype was achieved by biolistic-mediated transformation of the conventional soybean variety Jack with a 7.3-kb DNA fragment (i.e., SalI fragment) containing the coding sequences (i.e., 2mepsps and hppdPfW336) and required regulatory elements to express the 2mEPSPS and HPPD W336 proteins, respectively. Soybean calli from variety Jack were transformed using 10 μg of the SalI fragment and 6 bars of helium pressure. Successful transformants were selected on media containing diketonitrile (DKN), an HPPD inhibitor, and subcultured every 2 weeks until appeared. Putative transgenic clumps were amplified for 1 month. A 10-day selection process using DKN allowed for confirmation of the transformation. Green calli were matured for 2 months. Upon germination, root and shoot elongation, developed plantlets were transferred to a greenhouse and allowed to flower and set seed. From this selection process, double herbicide soybean event FG72 was selected.

3. Characterization of the Modified Plant

Southern blot analysis and DNA sequencing of double herbicide soybean event FG72 demonstrated the insertion of a DNA insert at a single locus consisting of two partial 3′histronAt sequences in a head to head orientation, followed by two complete copies of the SalI fragment in a head to tail orientation. Insertion of the transgenic DNA insert into the soybean genome induced the translocation of a genomic region to a new position directly downstream of the DNA insert. The translocated genomic region is joined at the 3′ junction by 158 nucleotide bases of the Ph4a748 promoter sequence. Analysis confirmed the absence of backbone sequence of the plasmid from which the SalI fragment was derived.

Bioinformatics analyses were performed on the transgenic DNA insert and flanking genomic sequences. Based on current databases and tools, no known open reading frames (ORFs) were interrupted due to the insertion of the transgenic DNA insert in the pre-insertion locus of the parent soybean Jack genome. The analyses also indicate that it is highly unlikely that any predicted newly created ORFs will lead to the expression of an unintended protein. An elongated transcript of the second copy of the 2mepsps gene is predicted but is not detected by western blot analysis.

Generational stability of the transgenic DNA insert was determined across multiple generations of the double herbicide tolerant soybean event FG72. Genomic DNA from three non-consecutive generations was analyzed by Southern blot analysis and the presence of the DNA insert was confirmed in each generation. Inheritance of the herbicide tolerance traits in double herbicide tolerant soybean event FG72 was assessed by measuring tolerance of the plants to the herbicide glyphosate. The herbicide tolerance traits were shown to segregate according to Mendelian rules of inheritance for a single genetic locus.

4. Product Information

Double herbicide tolerant soybean event FG72 differs from its conventional counterpart by the addition of the 2mepsps and hppdPfW336 coding sequences and their required regulatory elements. The insertion of the 2mepsps gene results in the expression of the 2mEPSPS protein: a 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) derived from the native maize (Zea mays L.) protein that contains two amino acid substitutions, rendering the enzyme insensitive to the herbicide glyphosate. As the safety of the 2mEPSPS protein was previously assessed by Health Canada and the CFIA in their evaluation of GlyTol™ cotton event GHB614, Bayer CropScience Inc. did not provide additional studies to support the safety of this protein. Supportive data was provided to demonstrate equivalence between the 2mEPSPS protein produced in double herbicide tolerant soybean event FG72 and the 2mEPSPS protein produced in an Escherichia coli expression system. The safety of the 2mEPSPS protein was previously demonstrated using the microbial-produced 2mEPSPS protein. Equivalency was established based on comparisons of the proteins' molecular weights, immunoreactivities, amino-terminal sequences, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry profiles and functional activities.

The insertion of the hppdPfW336 gene results in the expression of the HPPD W336 protein: a p-hydroxyphenylpyruvate dioxygenase derived from the Gram-negative bacterium Pseudomonas fluorescens that contains a single amino acid substitution (i.e., glycine substituted to tryptophan). This amino acid substitution renders the mutant HPPD protein insensitive to HPPD inhibitor herbicides. Studies related to the safety of the HPPD W336 protein were conducted using HPPD W336 protein produced in an Escherichia coli expression system. The petitioner provided supportive data to demonstrate equivalence between the microbial-produced HPPD W336 protein and the HPPD W336 protein produced in double herbicide tolerant soybean event FG72. Equivalency was established based on comparisons of the proteins' molecular weights, immunoreactivities, amino-terminal sequences, matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry profiles and functional activities.

The 2mepsps gene expressed in double herbicide tolerant soybean event FG72 is controlled by a constitutive promoter. Samples of the plant tissues were collected at various growth stages from greenhouse grown plants in Belgium in 2009. Average 2mEPSPS protein expression levels in micrograms protein per gram dry weight tissue (μg/g/dwt) evaluated by enzyme-linked immunosorbent assay (ELISA) are as follows: 569 μg/g/dwt in V4 leaf, 437 μg/g/dwt in V6 leaf, 668 μg/g/dwt in V8 leaf, 211 μg/g/dwt in V4 stem, 117 μg/g/dwt in V8 stem, 32.5 μg/g/dwt in V4 root, 43.7 μg/g/dwt in V8 root, and 2.62 μg/g/dwt in seed.

The hppdPfW336 gene expressed in double herbicide tolerant soybean event FG72 is controlled by a constitutive promoter. Samples of the plant tissues were collected at various growth stages from greenhouse grown plants in Belgium in 2009. Average HPPD W336 protein expression levels in micrograms protein per gram dry weight tissue (μg/g/dwt) evaluated by ELISA are as follows: 38.4 μg/g/dwt in V4 leaf, 35.8 μg/g/dwt in V6 leaf, 27.2 μg/g/dwt in V8 leaf, 16.6 μg/g/dwt in V4 stem, 6.04 μg/g/dwt in V8 stem, 5.81 μg/g/dwt in V4 root, 6.42 μg/g/dwt in V8 root, and 1.41 μg/g/dwt in seed.

5. Dietary Exposure

The consumption pattern of soybean-based food products is not expected to change with the addition of double herbicide tolerant soybean event FG72 to the food supply as it will be used in the same ways as currently commercialized soybeans.

The petitioner provided information regarding the estimated consumption of whole soybeans and the consumption of all kinds of oilseeds in various regional diets as reported by the Global Environmental Monitoring System (GEMS/Food) and was summarized in WHO, 2003. The European-type dietary estimate for whole soybeans was 0.1 g/person/day, since there was no estimate for whole soybeans, and the estimate for predicted daily intake of all kinds of oilseeds was 3.1 g/person/day. Using the highest 2mEPSPS protein expression levels measured in double herbicide tolerant soybean event FG72 seeds measured in micrograms protein per gram fresh weight tissue (fwt) (2.45 μg/g fwt), the predicted daily intake of 2mEPSPS protein from whole seeds was 0.245 μg/person/day and using the intake for all kinds of oilseeds it was 7.60 μg/person/day. For the highest HPPD W336 protein expression levels measured in double herbicide tolerant soybean event FG72 seeds measured in micrograms protein per gram fresh weight tissue (fwt) (1.26 μg/g fwt), the predicted daily intake of HPPD W336 protein from whole seeds was 0.126 μg/person/day and using the intake for all kinds of oilseeds it was 3.91 μg/person/day.

Since other types of soybean products are further processed, the protein expression levels for dry weight tissue were used to calculate exposure to 2mEPSPS and HPPD W336 proteins from other soybean products. Based on the USDA's Continuing Survey of Food Intakes by Individuals (US CSFII, 1994-96, 98), an estimate of total consumption of 287.8 g/kg body weight (bw)/day was assumed for infant formula (i.e., 0-6 months, 95th percentile) and was used as a conservative estimate for soybean-based infant formula intake. Infants consume the highest amount of soybean protein on a per kg basis of any age group. The highest 2mEPSPS protein expression levels measured in double herbicide tolerant soybean event FG72 seeds would give a predicted 1649 μg/kg bw/day 2mEPSPS protein intake for infants. The highest HPPD W336 protein expression levels measured in double herbicide tolerant soybean event FG72 seeds would give a predicted 402.92 μg/kg bw/day HPPD W336 protein intake for infants. An estimate of food intake of soybean-based products for adults (including soy powder, soy beverage, and soy yogurt but not including tofu) was 4.74 g/kg bw/day based on The Canadian Community Health Survey data (Statistics Canada, 2004) and summarized in a Chemical Health Hazard Assessment Division (CHHAD) 2008 food intake document (Health Canada). For adults, the highest 2mEPSPS protein expression levels measured in double herbicide tolerant soybean event FG72 seeds would give a predicted 27.2 μg/kg bw/day 2mEPSPS protein intake and using the highest HPPD W336 protein expression levels measured in double herbicide tolerant soybean event FG72 seeds would give a predicted 6.64 μg/kg bw/day HPPD W336 protein intake.

6. Nutrition

The compositional equivalence of double herbicide tolerant soybean event FG72 (sprayed with glyphosate and HPPD inhibitor herbicides, and unsprayed) to an unmodified control (Jack) and three conventional soybean varieties was evaluated through data generated from field trials conducted at 10 sites representative of major soybean growing areas located in the United States during the 2008 growing season.

Nutrients and/or anti-nutrients were analyzed in seed including: proximates, minerals, vitamins, amino acids, fatty acids, phytic acid, raffinose, stachyose, trypsin inhibitors, and lectin. Statistically significant differences were noted in 24 analytes; however, all values were within the range of natural variation for conventional soybeans. Based on the information provided by Bayer CropScience Inc., the nutritional composition of double herbicide tolerant soybean event FG72 is similar to conventional commercial soybean varieties.

7. Chemistry/Toxicology

Due to the technical difficulties of isolating sufficient amounts of both the 2mEPSPS and HPPD W336 proteins from double herbicide tolerant soybean event FG72 for the purposes of testing, the safety of the HPPD W336 protein was assessed using microbial-derived HPPD W336 protein. As the microbial-derived HPPD W336 protein was demonstrated to be equivalent to the HPPD W336 protein produced by double herbicide tolerant soybean event FG72, the test results using the microbial-derived protein are considered a valid indication of toxicity of the plant-produced HPPD W336 protein. The safety of the 2mEPSPS protein was previously assessed for Bayer CropScience Inc.'s GlyTol™ cotton event GHB614.

A 14-day acute oral toxicity study was conducted where groups of five young adult female Crl:OF1 mice were administered microbial-derived HPPD W336 protein by gavage at a dose of 1880 mg/kg bw or an equal amount of bovine albumin serum (BSA) as a control protein. Animals were observed daily for mortality and clinical signs of toxicity, but no deaths or treatment-related effects were observed. Based on these results, the acute oral LD50 of microbial-derived HPPD W336 protein in female Crl:OF1 mice was determined to be greater than 1880 mg/kg bw.

A 28-day sub-chronic oral toxicity study was also conducted, where groups of ten (i.e., 5 males and 5 females) C57BL/6J strain mice were administered by gavage either microbial-derived HPPD W336 protein or the control protein (BSA). HPPD W336 protein was administered at a dose of 940 mg/kg bw/day. Animals were observed daily for mortality and clinical signs of toxicity. A detailed physical examination was performed once during the acclimatization phase and at least weekly during the treatment period. Ophthalmological examinations were performed on all animals once during the acclimatization phase and at study termination. Blood samples were taken on Study Day 22 for standard haematology parameters and on the day of necropsy for standard clinical chemistry parameters. All animals were subject to necropsy, selected organs weighed and a standard set of tissues were fixed and examined microscopically. No treatment-related changes were observed in mice treated with microbial-derived HPPD W336 protein, with the exception of slightly lower mean aspartate and alanine aminotransferase activities noted for the clinical chemistry in males, which were statistically significant when compared to the controls. However, these changes were not considered toxicologically relevant based on the marginal decreases, which were observed in one sex only and with no concomitant histopathological findings. Based on the results of this study, a No Observed Adverse Effect Level (NOAEL) could be determined at a dose level of 940 mg/kg bw/day of HPPD W336 protein.

A comparison of amino acid sequence of the HPPD W336 protein to protein sequences of known toxins was conducted by the petitioner. The complete protein sequence was compared will all sequences available in the public reference databases (e.g., Uniprot-Swissprot) using the BLASTP program. In addition to sequence matches with HPPD proteins from various sources (including Pseudomonas fluorescens), an identity of 54% was observed with the VLLY protein and an identity of 49-50% with legiolysins, also called ILLY. The VLLY protein is described as a hemolysin from Vibrio vulnificus, a pathogenic bacterium present in seawaters and able to infect humans who consume seafood. The ILLY protein, also a hemolysin, is from strains of Legionella pneumophilia, a human pathogenic bacterium that is the causative agent of legionellosis.

The HPPD family of proteins are found in bacteria, plants, vertebrates, invertebrates, and fungi as they are involved in tyrosine metabolism. The VLLY and ILLY proteins belong to this HPPD family of proteins. The similarity identified in sequence matches corresponds to conserved domains, which is indicated by alignment between VLLY, ILLY, and the HPPD W336 amino acid sequences. VLLY and ILLY protein expression is shown to be necessary but not solely sufficient to produce haemolytic activity in bacteria.

The petitioner concluded that the sequence homology between the HPPD W336 protein and the VLLY and ILLY proteins is unlikely to be relevant. The sequence match is due to conserved domains in proteins that possess HPPD enzymatic activity. These domains are conserved between all HPPD proteins. Direct haemolytic activity of these proteins (i.e., HPPD W336, VLLY, and ILLY) was not demonstrated, indicating that this homology is unlikely to be biologically relevant. This conclusion is supported by the observation that the high doses of HPPD W336 protein administered in the 14-day acute oral toxicity and the 28-day sub-chronic oral toxicity studies (i.e., 1880 and 940 mg/kg bw, respectively) did not provide any evidence of haemolytic activity. On this basis, it was concluded that the homology between the HPPD W336 protein and the VLLY and ILLY proteins does not pose a health concern.

A heat stability study was submitted for the microbial-derived HPPD W336 protein. Based on the results of the study, the HPPD W336 protein is structurally heat-stable up to 60 minutes at 90 °C, but it is not functionally heat-stable. Functional activity of the HPPD W336 protein dropped below 50% after the protein was incubated at 45 °C for 20 minutes. At more elevated temperatures (i.e., 60 and 90 °C) functional activity is lost completely after 2.5 minutes.

Two simulated gastric fluid (SGF) digestibility studies were submitted by the petitioner for review. Only one study was considered in this assessment as it was performed in accordance with the principles of Good Laboratory Practice (GLP), where the test material was clearly identified. The results of that study showed that the HPPD W336 protein was not detectable in SGF containing pepsin at 30 seconds or at subsequent incubation times. A simulated intestinal fluid (SIF) digestibility was also submitted for review. This study showed that the HPPD W336 protein was rapidly degraded in SIF with pancreatin, with no residual protein bands visible, after 30 seconds of incubation.

The heat stability and digestibility studies provided by the petitioner strongly indicate that the HPPD W336 protein is unlikely to be a toxin or an allergen.

Based on the predicted levels of dietary exposure to the 2mEPSPS protein and the results an acute oral toxicity in mice assessed in the GlyTol™ cotton event GHB614 assessment, the margin of exposure (MOE) for 2mEPSPS protein ranges from 1.21×10⁵ to 5.71×10⁸ depending on the various intakes of 2mEPSPS protein.

Based on the predicted levels of dietary exposure to the HPPD W336 protein and the results of the 28-day sub-chronic oral toxicity study in mice, the MOE for the HPPD W336 protein ranges from 2.33×10³ to 5.22×10⁸ depending on the various predicted intakes of HPPD W336 protein.

Based on the currently available data, potential exposure to the 2mEPSPS protein and the HPPD W336 protein from double herbicide tolerant soybean event FG72 would not be a toxicological concern.

8. Allergenicity

A comparison of amino acid sequence of the HPPD W336 protein to protein sequences of known allergens was conducted. The three in silico approaches used in the study were: an overall sequence identity search (with a positive match defined as the whole HPPD W336 amino acid sequence having ³35% identity with a known allergen), an 80-amino acid sequence identity search (with a positive match defined as the 80-amino acid sequence having ³35% identity with a known allergen, and an 8-amino acid epitope search (with a positive match defined as the 8-amino acid sequence having 100% identity with a known allergen). All amino acid sequences derived from the HPPD W336 protein were compared against all known allergens present in the public allergen database, AllergenOnline, using the FASTA algorithm. Additionally, potential N-glycosylation sites in the HPPD W336 amino acid sequence were considered by searching for their known consensus sequence, which is often present in known allergens. According to the petitioner, there were no relevant sequence similarities (i.e. no positive matches identified) with known allergens. In addition, no potential N-glycosylation sites were identified in the amino acid sequence of the HPPD W336 protein.

The genetic modification to the parental Jack soybean genome did not result in an increase of endogenous soybean allergens in double herbicide tolerant soybean event FG72 compared to conventional soybean varieties, based on the expression level of known endogenous soybean allergens. Therefore, this modification does not pose an increased allergenic health risk than the parental soybean variety.

Based on the currently available data, potential exposure to the HPPD W336 protein from double herbicide tolerant soybean event FG72 would not be an allergenic concern.

Conclusion:

Health Canada's review of the information presented in support of the food use of double herbicide tolerant soybean event FG72 does not raise concerns related to food safety. Health Canada is of the opinion that food derived from double herbicide tolerant soybean event FG72 is as safe and nutritious as food from current commercial soybean varieties.

Health Canada's opinion deals only with the food use of double herbicide tolerant soybean event FG72. 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 environmental, animal, and human health safety of double herbicide tolerant soybean event FG72 with the intended use in animal feed. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from double herbicide tolerant soybean event FG72 destined for commercial sale.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

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For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca