Decision Document DD 2013-96: Determination of the Safety of Syngenta Canada Inc.’s Corn (Zea mays L.) Event 5307

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This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document BIO1994-11 - The Biology of Zea mays (L.) (Maize), and Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate, has evaluated information submitted by Syngenta Canada Inc. This information is in regard to the insect resistant trait in corn event 5307. The CFIA has determined that this plant with a novel trait (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to currently commercialized corn varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of Syngenta Canada Inc. corn event 5307 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of February 21, 2013. Any corn lines derived from corn event 5307 may also be released into the environment and used as livestock feed, provided that (i)  no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn in Canada, in terms of their potential environmental impact and livestock feed safety, and (iv) the novel genes are expressed at levels similar to those of the authorized line.

Additionally, with respect to its unconfined release into the environment, cultivation of corn event 5307 is subject to insect resistance management requirements.

Corn event 5307 is subject to the same phytosanitary import requirements as its unmodified counterparts. Corn event 5307 is required to meet the requirements of other jurisdictions; including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

Please note that the livestock feed and environmental safety assessments of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.

(publié aussi en français)

February 21, 2013

This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Animal Feed Division at:

1-800-442-2342
(613) 225-2342
59 Camelot Drive,
Ottawa Ontario K1A 0Y9

Table of Contents

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Corn event 5307, OECD Unique Identifier SYN-Ø53Ø7-1

Applicant: Syngenta Canada Inc.

Plant Species: Corn (Zea mays L.)

Novel Traits: Resistance to coleopteran pests including Diabrotica virgifera virgifera (Western corn rootworm) and Diabrotica longicornis barberi (Northern corn rootworm)

Trait Introduction Method: Agrobacterium-mediated transformation

Intended Use of the Modified Plant: Production of corn for livestock feed and human food. These plants are not intended to be grown outside the normal production area for corn in Canada.

II. Background Information

Syngenta Canada Inc. developed, through the use of recombinant deoxyribonucleic acid (DNA) techniques, a corn event resistant to a number of important coleopteran pests including Diabrotica virgifera virgifera (Western corn rootworm) and Diabrotica longicornis barberi (Northern corn rootworm). The corn event, designated as corn event 5307, was developed to provide a method to reduce yield losses from feeding damage caused by rootworm larvae.

Corn event 5307 was developed using recombinant DNA technology, resulting in the introduction of the engineered eCry3.1Ab gene and expression of the eCry3.1Ab insecticidal protein. eCry3.1Ab is a chimeric protein that contains regions of the mCry3A and Cry1Ab insecticidal proteins. The modified Cry3A (mCry3A) insecticidal protein is present in corn event MIR604, and a truncated version of Cry1Ab insecticidal protein is present in corn event Bt11 (4334 CB. 4374 CBR). Both corn event MIR604 and corn event Bt11 were previously authorized for unconfined environmental release and animal feed use in Canada (DD2007-68 and DD1996-12).

Corn event 5307 also contains the phosphomannose isomerase (pmi) gene from Escherichia coli (E. coli) for use as a selectable marker. This gene encodes the phosphomannose isomerase (PMI) enzyme. Expression of the PMI enzyme allows the plant to utilize mannose as a carbon source. This trait is of no agronomical interest but was used to select transformed plants during the development phase of corn event 5307.

Syngenta Canada Inc. has provided data on the identity of corn event 5307, a detailed description of the transformation method, data and information on the gene insertion site, gene copy number and levels of gene expression in the plant and the role of the inserted genes and regulatory sequences. The novel proteins were identified and characterized. Data were provided for the evaluation of the potential toxicity of the novel proteins to livestock and non-target organisms and potential allergenicity of the novel proteins to humans and to livestock.

Corn event 5307 was field tested at five and 12 test sites in 2007 and 2008, respectively. Some of the locations (Iowa, Illinois, Indiana, Minnesota, Missouri, Nebraska, South Dakota and Wisconsin) of these trials share similar environmental and agronomic conditions to corn production areas in Southern Ontario and Quebec and were considered representative of the major Canadian corn growing regions.

Agronomic characteristics of corn event 5307, including early emergence vigour, emerged plants, early growth vigour, early root lodging, snapped plants, ear height, plant height, plant with dropped ears, leaf colour, late season intactness, late root lodging, push test, stalk lodging, heat units to 50% pollen shed, heat units to 50% silking, plant population at harvest, barren plants, grain moisture, test weight, yield, susceptibilities to various corn pests and pathogens, dormancy/germination were compared to those of an unmodified corn control.

Nutritional components of corn event 5307, such as proximates, amino acids, fatty acids, vitamins, minerals, and anti-nutrients, were compared with those of an unmodified corn control. The Plant Biosafety Office of the Plant Health and Biosecurity Directorate, in conjunction with the Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, CFIA, have reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in the Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.

The PBRA Unit has considered:

  • the potential of corn event 5307 to become weeds of agriculture or be invasive of natural habitats;
  • the potential for gene flow from corn event 5307 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for corn event 5307 to become a plant pest;
  • the potential impact of corn event 5307 and its gene products on non-target species, including humans; and
  • the potential impact of corn event 5307 on biodiversity.

The Animal Feed Division (AFD), of the Animal Health Directorate, CFIA, has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed, as described in Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The AFD has considered both intended and unintended effects and similarities and differences between the modified plant and its counterpart relative to the safety and efficacy of feed ingredients derived from corn event 5307 for their intended purpose; including:

  • the potential impact of corn event 5307 on livestock nutrition; and
  • the potential impact of corn event 5307 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin, and worker/bystander exposure to the feed.

The AFD has also considered whether feeds derived from corn event 5307 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Syngenta Canada Inc. has provided the CFIA with a method for the detection and identification for corn event 5307.

III. Description of the Novel Traits

1. Development Method

The eCry3.1Ab and pmi genes were introduced in corn event 5307 via Agrobacterium-mediated transformation of immature embryos derived from a proprietary corn line. Embryos producing embryogenic callus were transferred to a cell culture medium containing mannose to select transformed cells. Corn event 5307 was identified as a successful transformant and was chosen for further development.

2. Resistance to Corn Rootworms (Diabrotica spp.)

Bacillus thuringiensis (B. thuringiensis) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, i.e. from Cry proteins. These Cry proteins are insecticidal to susceptible insect species after cleavage by proteases in the insect's gut, forming a protease-resistant active fragment that is the bio-active form of the proteins. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually result in cell lysis and insect death.

The mCry3A protein is a chimera of the Cry3A protein of B. thuringiensis subspecies tenebrionis, which possesses activity against corn rootworms, and the Cry1Ab protein of B. thuringiensis subspecies kurstaki, which possesses activity against some lepidopteran insects. The eCry3.1Ab protein was engineered by exchanging regions of the mCry3A and Cry1Ab proteins. The chimeric protein is only active against certain Diabrotica spp. and chrysomelid beetles and not against lepidopteran insects since the portion of the Cry1Ab protein used to create eCry3.1Ab is not from the region responsible for its lepidopteran activity.

The eCry3.1Ab gene is expressed in corn event 5307 using a constitutive promoter which confers ubiquitous expression of the eCry3.1Ab protein in corn. Tissue samples were collected at various growth stages from corn event 5307 hybrid plants grown at four representative United States (US) field trial sites. Levels of eCry3.1Ab protein were evaluated by enzyme-linked immunosorbent assays. Average eCry3.1Ab levels across all growth stages and sites, expressed in micro-grams eCry3.1Ab protein per gram dry weight tissue (µg/g dwt) ranged from 8 to 111 µg/g dwt in whole plant, <49 to 143 µg/g dwt in leaves, 9 to 43 µg/g dwt in roots, 4 to 6 µg/g dwt in kernels and were 0.10 µg/g dwt or less in pollen.

The eCry3.1Ab protein was shown to degrade readily in the soil. The eCry3A.1Ab protein was shown to lose its activity after 14 days of incubation in a loam soil when incorporated at a rate of 125 µg/g dwt of soil. Therefore the eCry3A.1Ab protein is unlikely to accumulate or persist in the soil.

The eCry3.1Ab protein expressed in corn event 5307 was purified from leaves and characterized. The identity of the purified protein was confirmed by Western immunoblot analysis, peptide mass mapping analysis and insecticidal activity.

The levels of eCry3.1Ab protein in corn event 5307 tissues were too low to extract sufficient amounts for evaluation of environmental and feed safety. To obtain sufficient quantities of eCry3.1Ab protein for safety studies, it was necessary to express the eCry3.1Ab gene in an E. coli production system. The equivalency of the plant-produced protein to the E. coli-produced protein was evaluated by comparing their molecular weight, immunological reactivity, insecticidal activity and glycosylation status. Based on the results, both proteins were found to be biochemically and functionally equivalent.

The potential mammalian toxicity and allergenicity of the eCry3.1Ab protein were evaluated. The eCry3.1Ab protein lacks sequence similarity to known mammalian toxins. No adverse effects were observed when the eCry3.1Ab protein was ingested by mice at a dose of 2000 milligrams protein per kilogram body weight (mg/kg bwt). The eCry3.1Ab protein lacks sequence similarity to known allergens. In vitro digestive fate studies showed that the protein is rapidly degraded in simulated gastric fluid, unlike protein allergens which are normally resistant to digestion. The eCry3.1Ab protein expressed in corn event 5307 is not glycosylated, unlike many allergens, providing additional evidence that the eCry3.1Ab protein expressed in corn event 5307 does not have the properties of known allergens.

Incubation for 30 minutes at 95°C or 65°C resulted in complete or partial inactivation of eCry3.1Ab protein, respectively. Collectively, these data indicate that the eCry3.1Ab protein is unlikely to be toxic to mammals or allergenic.

3. Expression of Phosphomannose Isomerase

The pmi gene from E. coli, which encodes the PMI enzyme, was introduced in corn event 5307. Corn cells producing PMI can utilize mannose as a primary carbon source, whereas cells lacking the pmi gene fail to proliferate in a mannose-based culture medium. Expression of the PMI protein is of no agronomic or industrial interest and was only used during the process of selecting transformants.

The PMI protein has been subject to previous CFIA safety assessment studies in corn events MIR162 (DD2010-79), 3272 (DD2008-70) and MIR604 (DD2007-68).
The pmi gene expressed in corn event 5307 is linked to a constitutive promoter. Tissue samples were collected at various growth stages from corn event 5307 hybrid plants grown at four representative US field trial sites. The PMI protein was detected at low levels in most plant tissues analyzed across all sites and growth stages.

Due to low expression of PMI protein in corn event 5307, it was not feasible to extract sufficient amounts of PMI protein from plant tissues for safety studies. It was necessary to express the pmi gene in an E. coli production system to obtain sufficient quantities of PMI protein. The equivalency of corn event 5307-produced PMI protein to the E. coli-produced PMI was evaluated by comparing their molecular weight, immunoreactivity, N-terminal amino acid sequence, glycosylation status and enzymatic activity. Based on the results, both proteins were found to be biochemically and functionally equivalent.

The potential toxicity and allergenicity of the PMI protein were evaluated. It is likely that small amounts of PMI proteins from various sources have always been present in the food and feed supplies due to the ubiquitous occurrence of PMI proteins in nature, including bacteria, yeast, some food plants and animals. PMI proteins are present in many mammalian tissues and in humans. Syngenta Canada Inc. investigated amino acid sequence homology of PMI protein produced in corn event 5307 with known protein toxins and found that the PMI protein does not possess significant amino acid sequence similarities to known toxins. Furthermore, no adverse effects were observed when the E. coli-produced PMI protein was ingested by mice at a dose of 2000 mg/kg bwt. Syngenta Canada Inc. investigated amino acid sequence homology of the PMI protein produced in corn event 5307 with known protein allergens. There is one region of sequence homology of eight contiguous identical amino acids between the PMI protein and a known allergen, alpha-parvalbumin, from Rana species. Further investigation using sensitive serum screening technology demonstrated that this sequence identity is not biologically relevant and has no implication for the potential allergenicity of the PMI protein. The PMI protein does not possess significant amino acid sequence similarities to other allergens. In vitro digestive fate studies have shown that the PMI protein is rapidly degraded in simulated gastric and intestinal fluids unlike protein allergens which are normally resistant to digestion. The PMI protein is inactivated following incubation at 65°C for 30 minutes.

Collectively, these data indicate that the PMI protein is unlikely to be a toxin or an allergen.

4. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that corn event 5307 contains one intact copy of the eCry3.1Ab and pmi gene cassettes inserted at a single site in the corn genome. No additional elements, including intact or partial DNA fragments of the eCry3.1Ab or pmi cassette or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in corn event 5307. Sequencing of the introduced DNA confirmed the sequence and organization of the genetic elements within the insert. A single nucleotide change was identified within a non-coding region of the insert; this change has no effect on the functionality of the insert. Sequencing also revealed that some truncation occurred at the right border and left border of the insert. These deletions have no effect on the functionality of the DNA insert and this phenomenon has been previously observed with Agrobacterium transformation.

The stability of the insert was demonstrated by Southern blot analysis over four generations of corn event 5307. In addition, concentrations of the eCry3.1Ab and PMI proteins in corn event 5307 tissues were measured across four generations and the results indicate stability of eCry3.1Ab and PMI protein expression across generations. The inheritance pattern of the eCry3.1Ab and pmi genes was analyzed by real-time polymerase chain reaction (PCR) across four generations and showed that both genes segregate according to the Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

1. Potential of Corn Event 5307 to Become a Weed of Agriculture or be Invasive of Natural Habitats.

The biology of corn, described in the CFIA biology document BIO1994-11 - The Biology of Zea mays L. (Maize), states that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Corn does not possess the potential to become weedy due to the lack of seed dormancy, the non-shattering nature of corn cobs and the poor competitive ability of seedlings. Syngenta Canada Inc. provided data on phenotypic and agronomic traits to establish that corn event 5307 is not weedier or more invasive than commercially available corn hybrids.

The CFIA evaluated data submitted by Syngenta Canada Inc. on the reproductive biology and life history traits of corn event 5307. This event was field tested in the US Corn Belt at five locations in the 2007 growing season and at 12 locations in the 2008 growing season. Some of the locations (Iowa, Illinois, Indiana, Minnesota, Missouri, Nebraska, South Dakota and Wisconsin) share similar environmental and agronomic conditions to corn production areas in Southern Ontario and Quebec and were considered representative of major Canadian corn growing regions. During the field trials, corn event 5307 was compared to an unmodified control. Phenotypic and agronomic traits were evaluated, covering a broad range of characteristics that encompass the entire life cycle of the corn plant. The traits included early emergence vigour, emerged plants, early growth vigour, early root lodging, snapped plants, ear height, plant height, plant with dropped ears, leaf colour, late season intactness, late root lodging, push test, stalk lodging, heat units to 50% pollen shed, heat units to 50% silking, plant population at harvest, barren plants, grain moisture, test weight, and yield. For the majority of the traits, no statistically significant differences between corn event 5307 and the unmodified control were observed. Although instances of statistically significant differences were observed between corn event 5307 and the unmodified control, there were no consistent trends in the data across locations or years that would indicate that any differences were due to the genetic modification. While corn event 5307 had a higher grain moisture, lower heat units to 50% pollen shed and higher plant height than that of the unmodified control in the 2007 growing season and a higher grain yield than that of the unmodified control in the 2008 growing season, the differences detected were small in magnitude and were not consistent over years or locations. The statistical analysis of these observations showed no biologically meaningful differences between corn event 5307 and the unmodified control, and supports a conclusion of phenotypic and agronomic equivalence to conventional corn.

Syngenta Canada Inc. provided information on the dormancy and germination of corn event 5307 seed under six different temperature regimes. During the trials, seed germination characteristics were evaluated, including percent germinated seed (normal and/or abnormal), percent hard seed, percent dead seed and percent firm swollen seed. Corn event 5307 was compared to an unmodified control and three commercial corn hybrids used as references. Although instances of statistically significant differences were observed between corn event 5307 and the unmodified control for five temperature regimes for percent germinated seed, the values for corn event 5307 were lower than that of the unmodified control and in majority were within the range of values established for the reference corn hybrids. Moreover, the lack of hard seed indicates that corn event 5307 does not possess seed dormancy.

Syngenta Canada Inc. provided information on the viability and morphology of pollen from corn event 5307. The viability, diameter and general morphology of pollen grains were evaluated. No statistically significant differences were detected between corn event 5307 and the unmodified control for any of the pollen characteristics evaluated.

In addition to the agronomic characteristics, susceptibility of corn even 5307 to various corn pathogens was evaluated (further detail provided below in Section 3: Altered Plant Pest Potential of Corn Event 5307).

The results showed that no competitive advantage was conferred to corn event 5307, other than resistance to corn rootworm, as the reproductive and growth characteristics of corn event 5307 were comparable to those of commercial corn hybrids. As feeding damage by rootworm larvae is not known to be a major factor restricting the establishment or distribution of corn in Canada, the introduction of this novel trait does not make corn event 5307 weedy or invasive of natural habitats.

The novel trait has no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that corn event 5307 has no altered weed or invasiveness potential in Canada compared to corn varieties currently grown in Canada.

2. Potential for Gene Flow from Corn Event 5307 to Sexually Compatible Plants Whose Hybrid Offspring May Become More Weedy of More Invasive

The biology of corn, as described in the CFIA biology document BIO1994-11 - The Biology of Zea mays L. (Maize), indicates that there are no wild relatives in Canada that can hybridize with corn. The rootworm resistance trait is unrelated to sexual compatibility and the information submitted by Syngenta Canada Inc. on corn event 5307 indicates that it behaves similarly to conventional corn in terms of reproduction.

The CFIA has therefore concluded that gene flow from corn event 5307 to wild relatives is not possible in Canada.

3. Altered Plant Pest Potential of Corn Event 5307

Corn is not considered a plant pest in Canada. The novel trait (resistance to corn rootworm) is unrelated to the potential for the plant to harbour new or increased populations of pathogens. Field evaluations of corn event 5307 confirmed the lack of any biologically meaningful change in susceptibility to pathogens (grey leaf spot, northern corn leaf blight, Southern corn leaf blight and eyespot disease) compared to an unmodified control grown at the same locations.

The unconfined environmental release of corn event 5307 without a proper management plan could potentially lead to the development of resistance to the eCry3.1Ab protein by its target pests. According to Syngenta Canada Inc., corn event 5307 is not intended to be cultivated as an individual event in Canada. If corn event 5307 is cultivated in Canada as a single event, an insect resistant management (IRM) plan addressing the conditions of the authorization must be implemented.

Thus, if used as intended, corn event 5307 will not display any altered plant pest potential compared to corn varieties currently grown in Canada.

4. Potential Impact of Corn Event 5307 on Non-Target Organisms

The novel eCry3.1Ab protein expressed in corn event 5307 is a chimeric protein containing amino acid sequences from the mCry3A and Cry1Ab Bt proteins. Both the mCry3A and Cry1Ab proteins have been shown to be safe to vertebrate species, including mammals, birds and fish (see DD2007-68, DD97-19, DD96-12). Syngenta Canada Inc. provided a detailed characterization of the eCry3.1Ab protein [as summarized in Part III: Description of the Novel Traits, Section 2. Resistance to Corn Rootworms (Diabrotica spp.)], which led to the conclusion that this protein is not a putative mammalian toxin or allergen. In addition, no detrimental effects were observed on juvenile bobwhite quails (Colinus virginianus) and mice over 14 days after ingestion of a single dose of 900 mg eCry3.1Ab protein/kg bwt or 2,000 mg eCry3.1Ab protein/kg bwt, respectively. Similarly, no detrimental effects were observed on survival and growth of juvenile channel catfish (Ictalurus punctatus) fed for 28 days using a fish diet containing 41% of corn event 5307 grain. Therefore, it is very unlikely that the eCry3.1Ab protein would be detrimental to vertebrate species, including human beings, interacting with corn event 5307 plants.

Typically the activity of Cry proteins is restricted to closely related insect species. Screening of insect species for sensitivity to the eCry3.1Ab protein showed that the activity spectrum of the eCry3.1Ab protein is restricted to some Coleopteran species of the Chrysomelidae family, including the Western corn rootworm (Diabrotica virgifera virgifera), the Northern corn rootworm (Diabrotica longicornis barberi), the Mexican corn rootworm (Diabrotica virgifera zea) and the Colorado potato beetle (Leptinotarsa decemlineata). The eCry3.1Ab protein is not active against the Southern corn rootoworm (Diabrotica undecimpunctata).

Five species of insects representing non-target insect species potentially exposed to the eCry3.1Ab protein expressed in corn event 5307 were tested in the laboratory for susceptibility to the eCry3.1Ab protein. The test insects were exposed for several days to several weeks to artificial diets containing concentrations of eCry3.1Ab that represent between 6.8 and 290 times very conservative estimates of the concentrations of eCry3.1Ab protein they could be potentially exposed to from cultivation of corn event 5307. The test insects were monitored for adverse effects on survival, development and, in some cases, fecundity. The following insects were tested: eggs, larvae, pupae and adults of the domestic honeybee (Apis mellifera), representing pollinators; larvae of the pink spotted ladybird (Coleomegilla maculata) representing above-ground predators and a non-target Coleoptera; nymphs of the flower bug (Orius laevigatus) representing above-ground predators; larvae of the carabid beetle (Poecilus cupreus) and adults of the rove beetle (Aleochara bilineata) representing soil-dwelling predators and a non-target Coleopteran species.

During these studies, no deleterious effects due to exposure to the eCry3.1Ab protein were observed on any of these test species, with the exception of minor effects on the development of P. cupreus. In the P. cupreus study, one to two day-old larvae were fed a diet containing 400 micrograms eCry3.1Ab protein per gram (µg/g) diet, until they reached the pre-pupal stage and then the emergence of the adults was monitored. This dose represents 62 times the worst-case eCry3.1Ab concentration ground-dwelling organisms could be exposed to from cultivation of corn event 5307. Control larvae were fed the same diet, without the eCry3.1Ab protein. There was no significant difference between the larval or pupal mortality of the treatment and control groups (8% and 10% mortality respectively). Adults that were treated with the eCry3.1Ab protein at the larval stage weighed on average 20% less than the control adults. P. cupreus larvae were tested under extreme eCry3.1Ab concentrations with no reduction of survival and minor effects on beetle weight. Therefore negative effects on P. cupreus and other ground beetles are likely to be negligible at field concentrations.  Currently there are no Coleoptera on the list of species at risk in Canada - PDF (653 kb) (http://www.cosewic.gc.ca/eng/sct0/rpt/rpt_csar_e.pdf). As the eCry3.1Ab protein is not active against lepidopteran species, larvae of the monarch butterfly (Danaus plexippus) will not be impacted by exposure to corn event 5307 pollen. No species at risk in Canada are expected to be harmed by cultivation of corn event 5307.

The earthworm Eisenia fetida was exposed for 14 days to the eCry3.1Ab protein incorporated in an artificial soil at a concentration representing approximately the worst-case eCry3.1Ab concentration earthworms could be exposed to from cultivation of corn event 5307. There were no effects on survival and weight of the earthworms exposed to the eCry3.1Ab protein compared to the control group. Therefore no negative effects on earthworms are anticipated from cultivation of corn event 5307.

Results of laboratory and field studies have shown that Cry proteins, like other proteins, are degradable in live soils, likely via enzymatic degradation by ubiquitous soil proteases. Live soil characterized as loam and collected from a corn-growing region in Indiana was used to assess the rate at which the eCry3.1Ab protein loses its bioactivity in soil. The results from this study showed that the eCry3.1Ab protein lost its bioactivity after a 14-day incubation in the soil. The rapid loss of biological activity in the test soil indicates that the accumulation of eCry3.1Ab protein in soil is unlikely and the persistence of active eCry3.1Ab protein in soil will likely be brief.

A potential route of exposure of aquatic organisms to eCry3.1Ab protein is through corn event 5307 pollen deposited into water bodies adjacent to corn event 5307 fields. However the low concentration of eCry3.1Ab in pollen along with the limited dispersal of corn pollen and likely eCry3.1Ab protein degradation in water suggest minimal levels of the eCry3.1Ab protein in water from corn event 5307 pollen. The transfer of corn event 5307 material into waterways surrounding fields may occur, however it is unlikely to result in ecologically significant concentrations of the eCry3A.1Ab protein in water bodies. The mean concentration of eCry3A.1Ab protein in corn event 5307 tissues is low at senescence, the developmental stage from which post-harvest plant debris will originate. Degradation of the eCry3.1Ab protein is likely to occur between harvest and the transfer of plant material into water bodies. In addition, the persistence of active eCry3.1Ab protein in soil following cultivation of corn event 5307 will likely be brief, suggesting minimal transfer of active eCry3.1Ab protein released in soil into water bodies. Collectively, these information elements indicate that significant exposure of aquatic organisms to the eCry3.1Ab protein is unlikely. Nevertheless a bioassay was conducted using a freshwater shrimp (Gammarus fasciatus) as representative freshwater crustacean. The shrimps fed for 5 days on whorl-stage leaf dics of corn event 5307, which represents a worse-case exposure scenario for freshwater invertebrates. No decreased survival was observed for the shrimps that had fed on corn event 5307 leaf discs compared to the control shrimps that had fed on non-transgenic leaf discs. The amount of leaf material consumed was similar in both groups. Therefore, the limited expected exposure of freshwater organisms to the eCry3.1Ab protein along with the lack of toxicity of the eCry3.1Ab protein on fish and crustaceans indicate that cultivation of corn event 5307 is unlikely to cause detrimental effects on freshwater organisms.

The safety of the PMI protein on non-target organisms has been previously established (see DD2007-68, DD2008-70, DD2010-79).

A detailed compositional analysis showed that grain and forage from corn event 5307 plants contain similar levels of nutrients and anti-nutrients compared to conventional corn varieties, indicating that the genetic modification did not cause an unintended change in the composition of corn event 5307.

Field evaluations of corn event 5307 showed that corn event 5307 did not display any increased resistance to pathogens compared to an unmodified control (See 3. Altered Plant Pest Potential of Corn Event 5307).

Collectively, these information elements indicate that no negative impacts on non-target organisms resulting from exposure to corn event 5307 plants are expected.

The CFIA has therefore determined that the unconfined release of corn event 5307 will not result in altered impacts on non-target organisms, including humans, compared to corn varieties currently grown in Canada.

5. Potential Impact of Corn Event 5307 on Biodiversity

Corn event 5307 is not expected to cause negative effects on non-target organisms and does not present altered weediness, invasiveness or plant pest potential. Since corn has no wild relatives with which it can outcross in Canada, there will be no transfer of the novel traits to other species in unmanaged environments. The novel traits are not expected to expand the range of cultivation of corn in Canada.

At present, crop rotation, the use of corn rootworm resistant corn varieties and chemical insecticide treatments are common practices to control corn rootworm in Canada. Corn event 5307 provides an alternative method to existing methods to control corn rootworm. Therefore, the reduction in local corn rootworm populations as a result of the release of corn event 5307 does not present a significant change from existing agricultural practices.

The CFIA has concluded that the novel genes and their corresponding traits do not confer to corn event 5307 any characteristic that would result in unintended environmental effects following unconfined release. The CFIA has therefore concluded that the potential impact on biodiversity of corn event 5307 is unlikely to be different from that of corn varieties currently grown in Canada.

6. Potential for Development of Insect Resistance to Corn Event 5307

Additionally, with respect to its unconfined release into the environment, cultivation of corn event 5307 is subject to insect resistance management requirements. According to Syngenta Canada Inc., corn event 5307 is not intended to be cultivated as an individual event in Canada. If corn event 5307 is cultivated in Canada as an individual event, an Insect Resistance Management plan must be implemented in order to delay the development of resistance to eCry3.1Ab in corn rootworm populations.

V. Criteria for the Livestock Feed Assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from corn event 5307, including the presence of gene products, residues, and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin, and worker/bystander exposure to the feed; and whether feeds derived from corn event 5307 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Corn Event 5307 on Livestock Nutrition

Nutrient and Anti-Nutrient Composition

The nutritional equivalence of corn event 5307 to an unmodified control was determined from replicated trials at six US sites during the 2008 growing season. At each location, corn event 5307 and the unmodified control were planted in a randomized complete block design, with three replicates (plots) for each corn type. Forage and seed samples were harvested from each plot at each location. Samples were pooled to create composite samples for each plot, from which sub-samples were obtained and shipped to Syngenta Crop Protection, Inc. (Greensboro, NC.). Sub-samples were ground and stored at –20°C, until composition analyses were conducted. Forage and grain samples were analysed for proximate (crude fat, ash, protein and moisture), acid detergent fibre (ADF), neutral detergent fibre (NDF), calcium and phosphorus. Seed samples were further analysed for amino acids, fatty acids, vitamins, minerals, secondary metabolites (ferulic acid, p-coumaric acid, inositol, furfural and raffinose) and anti-nutrients (phytic acid and trypsin inhibitor) as recommended by the Organization for Economic Co-operation and Development (OECD) consensus document for new varieties of corn (OECD, 2002)Footnote 1. Composition data was analysed statistically using a mixed model analysis of variance and statistical differences among corn types were identified (P<0.05). The composition data was also assessed within the natural variability of composition of conventional corn varieties, published in the International Life Sciences Institute (ILSI) Crop Composition Database (2008)Footnote 2.

No statistically significant differences were observed between corn event 5307 and unmodified control corn forage for proximate, carbohydrates, ADF and NDF across all locations. All means were within the range for conventional corn varieties literature valuesFootnote 2. For seed samples, there were no statistically significant differences between corn event 5307 and the unmodified control for proximate, carbohydrates, ADF, NDF, total dietary fibre (TDF), starch and minerals across all locations. All means were within the range for conventional corn varieties literature valuesFootnote 2.

Statistically significant differences were observed between corn event 5307 and the unmodified control for vitamins A, B6 and B9, however the mean differences were small and mean values of corn event 5307 were within the range of conventional corn varieties' literature valuesFootnote 2. Amino acids analysed in corn event 5307 and the unmodified control were not significantly different. Palmitic, stearic, linolenic and eicosenoic acids content in corn event 5307 were statistically significantly different compared to levels in the unmodified control, however the means were within the range of the conventional corn varieties' literature valuesFootnote 2 and therefore the differences were not considered biologically relevant. Furfural levels were below the level of quantification in both corn event 5307 and the unmodified control. No statistically significant differences were observed between corn event 5307 and the unmodified control for ferulic acid, p-coumaric acid, inositol, phytic acid, trypsin inhibitor and raffinose. All means were within the natural variation of conventional corn varieties literature valuesFootnote 2.

Broiler Performance

A total of 900 birds were randomly distributed into 60 pens (15 birds per pen) to evaluate the performance of birds on diets prepared with corn event 5307 compared to those prepared from the unmodified control and a conventional corn variety (NCSU 2007). The poultry diets were formulated based on the individual nutrient analysis for each of the grain sources to meet the nutritional requirements for chickens. Corn grain from each variety was incorporated at 52-64% in the diets for the starter, grower and finisher diets during a 49-day trial. Water and feed were provided to the birds on a continual basis. Parameters evaluated during the study included broiler mortality, body weight, feed intake feed conversion and carcass yield. Statistically significance difference between corn treatments diets were determined (P<0.05).

The overall broiler survival was about 98% for both males and females at the end of the study period, with no effect of corn variety on survival at any time of the study. There were no statistically significant differences between birds on corn event 5307 diets and those on the unmodified control diet for body weights, weight gains and feed consumption over the 49-day period. Broilers fed corn event 5307 diets had improved feed conversion during the grower period (day 16-35 and day 0-35), when compared to broilers on the unmodified control diets, but no statistically significant differences were observed during the day 0-49 period. No statistically significant differences were observed between broilers on corn event 5307 diets and unmodified control for all carcass characteristics measured. Broiler chickens fed diets prepared with corn event 5307 grain did not show any adverse effects on health or growth performance when compared to chickens fed diets prepared with either the unmodified control grain or the conventional corn variety.

Conclusion

It was concluded, based on the evidence provided by Syngenta Canada Inc., that the nutritional composition of corn event 5307 is similar to conventional corn varieties. No biologically relevant differences were observed on the performance and health of birds consuming diets containing corn event 5307 grain.

2. Potential Impact of Corn Event 5307 on Animal Health andHuman Safetyas it Relates to the Potential Transfer of Residues into Foods of Animal Origin, and Worker/Bystander Exposure to the Feed

Corn event 5307 is resistant to some coleopteran pests as a result of insertion of genes encoding the chimeric protein eCry3.1Ab. The event also contains a gene coding for the marker protein PMI. The assessment of corn event 5307 evaluated the impact of the following potential hazards relative to the safety of feed ingredients derived from this event:

  • The presence of novel proteins PMI and eCry3.1Ab

Novel PMI and eCry3.1Ab proteins

The safety of PMI, included only as selectable marker, has already been characterized for three prior events; corn event MIR162 (DD2010-79), corn event 3272 (DD2008-70), and corn event MIR604 (DD2007-68). Syngenta Canada Inc. supplied data to verify the safety of PMI protein within corn event 5307. The PMI protein shares no significant biologically relevant sequence homology with any known toxins or allergens and lacks a mode of action that suggests that it is intrinsically toxic. The PMI protein is heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. No signs of toxicity were demonstrated in the single-dose oral toxicity studies in mice using purified PMI proteins from an E. coli over expression system at doses up to 2000 mg/kg bwt. These factors support the lack of intrinsic toxicity or allergenicity of the PMI protein. Furthermore, the PMI protein was previously evaluated and authorized in prior events in Canada.

The eCry3.1Ab protein has an insecticidal mode of action and does not have a history of safe use in livestock, although the mCry3A and truncated Cry1Ab proteins, from which the chimeric eCry3.1Ab protein is derived, are present in crops that have been approved by the CFIA. The eCry3.1Ab protein shares no significant biologically relevant sequence homology with any known toxins or allergens. This protein is also heat labile and rapidly degraded under conditions similar to those encountered in the gastrointestinal tract. No signs of toxicity were demonstrated in single-dose oral toxicity studies in mice using purified eCry3.1Ab protein from an E. coli over expression system at doses up to 2000 mg/kg bwt. These factors support the lack of intrinsic toxicity or allergenicity of the eCry3.1Ab protein, in respect of Animal Health and Human Safety.

Furthermore, the lack of performance effects or significant health observations in the broiler study using grain derived from corn event 5307 supports the safety of PMI and eCry3.1Ab proteins.

Conclusions

Feed ingredients derived from corn event 5307, are considered to meet present ingredient definitions for corn, and as such are approved for use as livestock feed in Canada.

VI. New Information Requirements

If, at any time, Syngenta Canada Inc. becomes aware of any new information regarding risk to the environment, including human health, or livestock feed use that could result from the release of corn event 5307, or lines derived from it, Syngenta Canada Inc. will immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of corn event 5307 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and environmental release authorization of corn event 5307.

VII. Regulatory Decision

Based on the review of the data and information submitted by Syngenta Canada Inc. and other relevant information, the CFIA has determined that corn event 5307 does not present altered environmental risk when compared to currently commercialized corn varieties in Canada.

Based on the review of submitted data and information by Syngenta Canada Inc. including comparisons of corn event 5307 with the unmodified control, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the novel gene and its corresponding traits will not confer to corn event 5307 any characteristic that would raise any concerns regarding the safety or nutritional composition of corn event 5307. Grain corn, its by-products and corn oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Corn event 5307 has been assessed and found to be as safe as and as nutritious as traditional corn varieties. Corn event 5307 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Unconfined release into the environment and use as livestock feed of corn event 5307 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of February 21, 2013. Any corn lines derived from corn event 5307 may also be released into the environment and used as livestock feed, provided that no inter-specific crosses are performed, the intended uses are similar, it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown corn in Canada, in terms of their potential environmental impact and livestock feed safety, and the novel genes are expressed at levels similar to those of the authorized line.

Additionally, with respect to its unconfined release into the environment, cultivation of corn event 5307 is subject to insect resistance management requirements.

Corn event 5307 is subject to the same phytosanitary import requirements as its unmodified counterparts. Corn event 5307 is required to meet the requirements of other jurisdictions; including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of corn event 5307.

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