DD2007-67: Determination of the Safety of Monsanto Canada Inc.'s Soybean (Glycine max (L.) Merr.) Event MON 89788

Issued: 2007-07

This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document BIO1996-10, "The Biology of Glycine max (L.) Merr. (Soybean)", and Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office, of the Plants Products Directorate, the Biotechnology Environmental Release Assessment Unit of the Science Strategies Directorate and the Animal Feed Division, has evaluated information submitted by Monsanto Canada Inc. This information is in regard to the tolerance of soybean event MON 89788 to glyphosate-based herbicides. 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 soybean varieties in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of soybean event MON89788 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division as of July 3, 2007. Any soybean lines derived from event MON 89788 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, and (iii) it is known based on characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybean in Canada, in terms of their potential environmental impact and livestock feed safety.

Soybean event MON 89788 is subject to the same phytosanitary import requirements as its unmodified counterpart.

Please note, that the livestock feed and environmental safety 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.

Table of Contents

I. Brief Identification of the Modified Plant

II. Background Information

III. Description of the Novel Trait

1. Tolerance to Glyphosate
2. Development Method
3. Stable Integration into the Plant Genome

IV. Criteria for the Environmental Assessment

1. Potential of Soybean event MON 89788 to Become a Weed of Agriculture or be Invasive of Natural Habitats
2. Potential for Gene Flow from Soybean event MON 89788 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
3. Altered Plant Pest Potential of Soybean event MON 89788
4. Potential Impact of Soybean event MON 89788 on Non-Target Organisms
5. Potential Impact of Soybean event MON 89788 on Biodiversity

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Soybean event MON 89788 on Livestock Nutrition
2. Potential Impact of Soybean event MON 89788 on Livestock and Workers/Bystanders

VI. New Information Requirements

VII. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Soybean Event MON 89788, OECD Unique Identifier MON-89788-1

Applicant: Monsanto Canada Inc.

Plant Species: Soybean (Glycine max (L.) Merr)

Novel Trait: Tolerance to glyphosate-based herbicides

Trait Introduction Method: Agrobacterium-mediated transformation

Proposed Use of the Modified Plant: Commercial production of soybean grain for human consumption and for livestock feed. These plants are not intended to be grown outside the normal cultivation area for soybean in Canada.

II. Background Information

Monsanto Canada Inc. developed, through the use of recombinant DNA techniques, a glyphosate-tolerant soybean. The soybean event, designated as MON 89788, was developed to provide an alternative method to control weeds in soybean production.

Soybean event MON 89788 was developed using recombinant DNA technology, resulting in the introduction of the bacterial gene cp4 epsps from Agrobacterium sp. strain CP4. This gene encodes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) enzyme, which has a much reduced affinity for glyphosate relative to endogenous soybean epsps enzyme, thereby conferring glyphosate- tolerance to soybean event MON 89788.

Monsanto Canada Inc. has provided data on the identity of soybean event MON 89788, 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 protein was identified and characterized. Data was provided for the evaluation of the potential toxicity of the novel protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans and to livestock.

Soybean event MON 89788 has been field tested in the United States and the data for trial year 2005 were submitted. Some of the locations of these trials share similar environmental and agronomic conditions to Southwestern Ontario and were considered representative of major Canadian soybean growing regions.

Agronomic characteristics of soybean event MON 89788 such as seed dormancy, vegetative vigour, time to maturity, flowering period, susceptibilities to various soybean pests and pathogens, pre-harvest seed loss characteristics and seed production were compared to those of unmodified soybean counterparts. Pollen characteristics and symbiotic interactions with the bacterium Bradyrhizobium japonicum were also evaluated.

Nutritional components of soybean event MON 89788, such as proximates, amino acids and fatty acids were compared with those of unmodified soybean counterparts.

The Plant Biosafety Office (PBO) of the Plant Health and Biosecurity Directorate, in conjunction with the Biotechnology Environmental Release Assessment (BERA) Unit of the Science Strategies 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), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". The BERA Unit has considered:

• potential of soybean event MON 89788 to become a weed of agriculture or be invasive of natural habitats;
• potential for gene flow from soybean event MON 89788 to wild relatives whose hybrid offspring may become more weedy or more invasive;
• potential for soybean event MON 89788 to become a plant pest;
• potential impact of soybean event MON 89788 or its' gene products on non-target species, including humans; and
• potential impact of soybean event MON 89788 on biodiversity.

The Animal Feed Division, 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 Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources". The Animal Feed Division has considered:

• potential impact of soybean event MON 89788 on livestock nutrition; and
• potential impact of soybean event MON 89788 on livestock and workers/bystanders

Monsanto Canada Inc. has provided the CFIA with a method for the detection and identification of soybean containing the soybean event MON 89788.

III. Description of the Novel Trait

1. Tolerance to Glyphosate

A gene derived from the Agrobacterium sp. strain CP4 (cp4 epsps) which imparts field level tolerance to glyphosate, the active ingredient in Roundup® brand herbicides, was introduced into soybean, and the transformation event was named MON 89788. A plant-derived coding sequence expressing an optimized chloroplast transit peptide was fused to the cp4 epsps coding sequence. This peptide facilitates the import of the newly translated epsps enzyme into the chloroplast, the site of amino acid biosynthesis. epsps is an enzyme involved in the shikimic acid metabolic pathway which is essential for the production of the aromatic amino acids. The native soybean epsps enzyme is sensitive to glyphosate. The herbicide disrupts the shikimic acid pathway, leading to growth suppression or death of the plant. The CP4 epsps version of this enzyme is expressed in soybean event MON 89788, and confers glyphosate tolerance since it continues to catalyze the production of aromatic amino acids in the presence of glyphosate due to a reduction in the binding of glyphosate to the CP4 epsps in comparison to the native soybean epsps.

The CP4 epsps protein produced in MON 89788 soybean is the same as the CP4 epsps protein produced in other Roundup Ready® crops which have already been approved for unconfined release and animal feed uses in Canada, including soybean event GTS 40-3-2 and Roundup Ready 2 corn (NK603).

The CP4 epsps protein has been subject previously to a number of safety assessment studies. To obtain sufficient quantities of CP4 epsps protein for evaluation of environmental and feed safety, it was necessary to express the cp4 epsps gene in an E. coli production system. The equivalency of the MON 89788 soybean-produced CP4 epsps protein to the E. coli-produced CP4 epsps was evaluated by comparing their molecular weights, immunoreactivity, glycosylation status, N-terminal sequence analysis, MALDI-TOF mass spectrometry and functional activity. Based on the results, the proteins were found to be equivalent.

Demonstration of protein equivalence between E. coli- and MON 89788-produced CP4 epsps proteins allows utilization of the existing data to confirm the safety of the CP4 epsps protein in MON 89788. Previous assessments have shown that the CP4 epsps protein did not cause any adverse effects in mice at a level of 572 mg/kg body weight, and that the CP4 epsps protein, unlike many allergens, is readily degraded in simulated mammalian gastric and intestinal fluids. CP4 epsps protein expressed in MON 89788 soybean is structurally and functionally similar to epsps enzymes present in many foods with a long history of safe use in Canada, and therefore would not be expected to be toxic or allergenic. In addition, Monsanto Canada Inc. provided an updated bioinformatic evaluation of the CP4 epsps protein which confirmed the lack of relevant similarities between the CP4 epsps protein sequence and sequences of known allergens.

The cp4 epsps gene expressed in soybean event MON 89788 is linked to a constitutive promoter. Samples of soybean tissues were collected at various growth stages from five representative US field trial sites. Average CP4 epsps protein expression in micro-grams protein per gram dry weight tissue (g/g dwt) as evaluated by enzyme-linked immunosorbent assay are as follows: 290-340 g/g dwt in leaf, 74 g/g dwt in root, 220 g/g dwt in forage and 150 g/g dwt in grain.

2. Development Method

The elite soybean variety A3244 was transformed with a binary plasmid vector carrying the cp4 epsps gene. The gene were introduced via Agrobacterium tumefaciens transformation into A3244 meristem cells. Transformants were selected based on tolerance to glyphosate. Event MON 89788 was identified as a successfully transformed event and was chosen for further development.

3. Stable Integration into the Plant Genome

Molecular characterization by Southern blot analysis demonstrated that soybean event MON 89788 contains one intact copy of the cp4 epsps gene cassette inserted at a single site in the soybean genome. Sequencing of the introduced DNA confirmed the sequence of the insert and the organization of the genetic elements. No additional elements, including intact or partial DNA fragments of the cp4 epsps cassette or backbone sequences, from the plasmid vector, linked or unlinked to the intact gene cassette, were detected in soybean event MON 89788. The presence of the cp4 epsps gene cassette and absence of backbone sequences in MON 89788 soybean was further confirmed by Southern blot analyses over four generations of the MON 89788 breeding tree. Segregation analyses across three generations were performed to determine the inheritance of the glyphosate tolerance trait. The results of the analyses are consistent with the finding of a single active site of insertion that segregates according to the Mendelian laws of genetics.

IV. Criteria for the Environmental Assessment

1. Potential of Soybean event MON 89788 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The biology of soybean, described in the CFIA Biology Document BIO1996-10, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Soybean does not possess the potential to become weedy due to the lack of seed dormancy and the poor competitive ability of seedlings.

Soybean event MON 89788 was tested in 17 locations in 2005 in the US. Some of these locations share similar environmental and agronomic conditions to Southwestern Ontario and were considered representative of major Canadian soybean growing regions. A total of 11 phenotypic characteristics were evaluated: seedling vigor, early stand count, days to 50% flowering, flower colour, lodging, pod shattering, final stand count, plant height, grain test weight, grain moisture and yield. The phenotypic characteristic data showed no biologically meaningful differences between MON 89788 soybean and the unmodified variety A3244, or a selection of commercially available soybean varieties, and support a conclusion of phenotypic equivalence to currently commercialized soybean varieties. The seed dormancy and germination of soybean event MON 89788 was compared with the unmodified control variety A3244. No significant differences were detected in percent germinated seed, percent dead seed, and percent viable firm swollen seed. Additionally, no viable hard seeds were detected in soybean event MON 89788 (viable hard seed is associated with seed dormancy).

No significant differences in percent viable pollen or average pollen diameter were detected between MON 89788 soybean and the A3244 control line.

Data on the susceptibility to a range of insect pest and disease, and response to abiotic stressors were also recorded at each of the 17 field trial sites. No increase or decrease outside of the reference range was observed in any insect, disease, or abiotic stressor in MON 89788 soybean.

The introduction of the glyphosate tolerance trait did not make MON 89788 soybean weedy or invasive of natural habitats since none of the soybean's reproductive or growth characteristics were modified and MON 89788's tolerance to abiotic and biotic stresses was unchanged. No competitive advantage was conferred to MON 89788 soybean, other than that conferred by tolerance to glyphosate herbicide. The emergence of MON 89788 soybean volunteer plants, should they arise, can easily be managed by mechanical means and other available chemicals currently used to control Roundup Ready soybeans.

The agronomic stewardship plan, which contains a herbicide tolerance management plan, submitted by Monsanto Canada Inc. was evaluated by the CFIA and determined to be satisfactory. The herbicide tolerance management plan includes recommendations on agricultural systems incorporating glyphosate tolerant soybean and provides an efficient mechanism for growers to report any agronomic problems with this product to Monsanto Canada Inc., facilitating the ongoing management of glyphosate tolerant soybeans.

In the longer term, the continued use any herbicide may increase selection pressure for the development of herbicide resistant weeds. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, Monsanto Canada Inc. will communicate essential elements of its stewardship plan to growers and agriculture extension personnel, in both private and public sectors, to promote recommended management practices, such as use of alternate control tools as appropriate to help minimize the development of resistant weed populations.

On the basis of this information, the CFIA concluded that the soybean event MON 89788 has no increased weediness or invasiveness potential compared to currently commercialized soybean varieties.

2. Potential for Gene Flow from Soybean event MON 89788 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of soybean, as described in CFIA Biology Document BIO1996-10, shows that soybeans exhibit a high percentage of self-fertilization and cross pollination is usually less than one percent, suggesting that any pollen flow from cultivated soybeans to related species is minimal. Natural hybridization between cultivated soybean and the wild annual species Glycine soja can occur. G. soja is not naturalized in North America, and although this species could occasionally be grown in research plots, there are no reports of its escape from such plots to unmanaged habitats.

The CFIA has therefore concluded that the potential for gene transfer from MON 89788 soybean to soybean relatives in managed ecosystems is negligible, and that there is no potential for gene transfer to related wild species in Canada.

3. Altered Plant Pest Potential of Soybean event MON 89788

The novel trait (tolerance to glyphosate) is unrelated to plant pest potential, and soybean is not considered a plant pest in Canada. Ecological evaluations of MON 89788 soybean did not show any increase or decrease in susceptibility to any insect or disease stressor that was outside of the reference range generated from commercial soybean varieties grown at the same locations. These stressors included 12 pest insects and 18 diseases.

The CFIA has, therefore, determined that the soybean event MON 89788 does not display any altered pest potential compared to currently commercialized soybean varieties.

4. Potential Impact of Soybean event MON 89788 on Non-Target Organisms

The safety of the CP4 epsps protein to non-target organisms, including humans, has previously been established. CP4 epsps protein expressed in MON 89788 soybean tissues is the same as the CP4 epsps protein produced in Roundup Ready soybean event GTS 40-3-2, which has a history of safe use in Canada.

Composition analyses showed that the levels of key nutrients and anti-nutrients in MON 89788 soybean seed and forage are comparable to those in commercial soybean varieties.

Ecological evaluations of MON 89788 soybean did not show any increase in resistance to insects or pathogens compared to commercial soybean varieties.

Ecological evaluations showed that the abundance of beneficial arthropods in MON 89788 soybean plots was equivalent to that in conventional soybean grown at the same locations. The beneficial organisms assessed included the seven spotted ladybeetle (Anatis septempunctata), green lacewing (Chrysoperla rufilabris), Nabis sp., Orius sp., and spiders.

Based on the above, the CFIA has determined that the unconfined release of soybean event MON 89788 will not result in altered impacts on interacting organisms, including humans, compared to current commercial soybean varieties.

5. Potential Impact of Soybean event MON 89788 on Biodiversity

Soybean event MON 89788 has no novel phenotypic characteristics that would extend its range beyond the current geographic range of soybean production in Canada. Since soybean has no wild relatives with which it can outcross in Canada, there will be no transfer of the glyphosate tolerance trait from MON 89788 soybean to plants in unmanaged environments.

MON 89788 soybean does not pose a risk to interacting non-target organisms. In addition, Monsanto Canada Inc. provided data showing that the introduction of the glyphosate-tolerance trait does not alter the symbiotic relationship between the bacterium Bradyrhyzobium japonicum and MON 89788 soybean compared to conventional soybean.

MON 89788 soybean provides an alternative method of weed control in soybean production. The use of broad spectrum herbicides has the intended effect of reducing local weed populations within agricultural fields and this may reduce local weed species biodiversity, and possibly other trophic levels which utilize these weed species. It must be noted, however, that reduction in weed biodiversity in agricultural fields is not unique to the use of plants with novel traits, and is a common practice in virtually all modern agricultural systems.

The CFIA has therefore concluded that the potential impact on biodiversity of soybean event MON 89788 is equivalent to that of currently commercialized soybean varieties.

V. Criteria for the Livestock Feed Assessment

1. Potential Impact of Soybean event MON 89788 on Livestock Nutrition

Nutritional Composition and Anti-Nutritional Factors

The compositional equivalence of MON 87988 to its isogenic control A3244 was assessed from five replicated sites in the US. Whole forage and seed samples were analyzed for proximate, ADF, NDF, amino acids, fatty acids, Vitamin E and isoflavones (daidzein, glycitein and genistein). No statistically significant differences were observed between MON 89788 and A3244 forage for fat, ash, protein, ADF and NDF, except for moisture. All means were within the tolerance interval generated for commercial reference soybean and also within literature values. In the combined-site analyses, there were no statistically significant differences between MON 89788 and A3244 grain for ash, fat, protein, ADF and NDF. No statistically significant differences were observed between MON 89788 and A3244 grain for all amino acids measured. All means were within the tolerance interval of the commercial reference soybean. No statistically significant differences were observed between MON 89788 and A3244 grain for all fatty acids analyzed across locations. All means were within the commercial tolerance interval. Vitamin E was statistically significantly higher in MON 89788 compared to A3244 grain in the combined-site analyses, however no statistically significant differences were observed within sites, and all means were within the commercial tolerance interval and literature values. Diadzein and glycitein were statistically significantly lower in MON 89788 than A3244 grain, while no statistically significant differences were observed between MON 89788 and control for genistein. No consistent significant differences were observed within sites for the isoflavones. All means were within the commercial tolerance interval and also within the variation of literature values.

The evidence provided by Monsanto Canada Inc. supports the conclusion that the nutritional composition of soybean line MON 89788 is substantially equivalent to its isogenic control A3244.

Anti-Nutrients

Phytic acid, trypsin inhibitor, lectin, raffinose and stachyose were analyzed in MON 87988 grain and compared to A3244 control. The levels of raffinose and stachyose were statistically significantly lower in MON 89788 than A3244 in one site, while raffinose was statistically higher in the test than control in another site. No statistically significant differences were however observed between MON 87988 when compared to A3244 grain for trypsin inhibitor, phytic acid, lectin, raffinose and stachyose across locations. All means were within the conventional tolerance interval and also within literature values.

2. Potential Impact of Soybean event MON 89788 on Livestock and Workers/By-standers

epsps is an enzyme present in many feeds with a long history of safe use in Canada, and therefore would not be expected to be toxic or allergenic. The CP4 epsps enzyme is from Agrobacterium strain CP4, a soil bacterium, which is not a known human or animal pathogen. The amino acid sequence of the CP4 epsps protein found in event MON 89788 is identical to the CP4 epsps protein in Roundup Ready crops previously approved in Canada. CP4 epsps shares no biologically relevant significant homology with known toxins or allergens, it is present in small amounts in the feed, it is heat labile and it is rapidly degraded under the conditions present in the gastrointestinal tract.

An acute mouse gavage study demonstrated that the CP4 epsps is not toxic. No treatment-related adverse effects were observed in animals administered CP4 epsps protein by oral gavage at doses up to 475 mg/kg body weight.

Based on the predicted exposure levels and the results of the above tests, no significant risk to livestock and workers/by-standers is expected from exposure to the CP4 epsps protein in soybean event MON 89788.

VI. New Information Requirements

If at any time, Monsanto Canada Inc. becomes aware of any information regarding risk to the environment, including risk to human or animal health, that could result from release of soybean event MON 89788 materials in Canada or elsewhere, Monsanto 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 event MON 89788 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of soybean event MON 89788.

VII. Regulatory Decision

Based on the review of the data and information submitted by Monsanto Canada Inc., and through comparisons of soybean event MON 89788 with unmodified soybean counterparts, the Biotechnology Environmental Release Assessment Unit of the Science Strategies Directorate, CFIA, has concluded that the novel gene and its corresponding trait does not confer to soybean event MON 89788 any characteristic that would result in unintended environmental effects following unconfined release.

Based on the review of submitted data and information by Monsanto Canada Inc., including comparisons of soybean event MON 89788 with its unmodified soybean counterparts, the Animal Feed Division has concluded that the introduced gene and its corresponding trait will not confer to soybean event MON 89788 any characteristic that would raise any concerns regarding the safety or nutritional composition of soybean event MON 89788. Soybean grain, its byproducts and soybean oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Soybean event MON 89788 has been assessed and found to be as safe as and as nutritious as traditional soybean varieties. Soybean event MON 89788 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of soybean event MON89788 is therefore authorized by Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division as of July 3, 2007. Any soybean lines derived from event MON 89788 may also be released into the environment and used as livestock feed, provided no inter-specific crosses are performed, provided the intended uses are similar, and provided it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybean in Canada, in terms of their potential environmental impact and livestock feed safety.

Soybean event MON 89788 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of soybean event MON 89788.