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Decision Document DD95-05:
Determination of Environmental Safety of
Monsanto Canada Inc.'s Glyphosate Tolerant
Soybean (Glycine max L.) Line GTS 40-3-2

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and the guidelines Dir95-03 Guidelines for the Assessment of Plants with Novel Traits as Livestock Feed.

The Canadian Food Inspection (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division and the Feed Section of the Animal Health and Production Division, has evaluated information submitted by Monsanto Canada Inc. regarding the soybean line GTS 40-3-2. This line has a Roundup-Ready™ gene that expresses novel tolerance to glyphosate, the active ingredient of Roundup® herbicide. CFIA has determined that these plants do not present altered environmental interactions when compared to currently commercialized soybean varieties in Canada and are considered substantially equivalent to soybean currently approved as livestock feed.

Unconfined release into the environment, including feed use of line GTS 40-3-2, and other Glycine max lines derived from it, but without the introduction of any other novel traits, is therefore considered safe.

Table of Contents

1. Brief Identification of the Plant with Novel Traits (PNT)
2. Background Information
3. Description of the Novel Trait
   1. Glyphosate tolerance
   2. Development Method
   3. Stable Integration into the Plant's Genome
4. Assessment Criteria for Environmental Safety
   1. Potential of the PNT to Become a Weed of Agriculture or be Invasive of Natural Habitats
   2. Potential for Gene Flow to Wild Relatives whose Hybrid Offspring may Become more Weedy or more Invasive
   3. Altered Plant Pest Potential
   4. Potential Impact on Non-Target Organisms
   5. Potential Impact on Biodiversity
5. Nutritional Assessment Criteria as Livestock Feed
   1. Anti-Nutritional Factors
   2. Nutritional Composition of the PNT
   3. Feeding Trials
6. Regulatory Decision

I. Brief Identification of the Plant with Novel Traits (PNT)

II. Background Information

Monsanto Canada Inc. has developed a soybean line tolerant to glyphosate, the active ingredient of the broad-spectrum non-residual herbicide Roundup®. This soybean line, referred to as GTS 40-3-2 in the present document, will allow the use of Roundup® as a post-emergence herbicide, thus providing an alternative for weed control in soybean production.

The development of GTS 40-3-2 was based on recombinant DNA technology, through the introduction of a bacterial gene into the soybean commercial cultivar "A5403". This gene imparts reduced sensitivity to Roundup® at the biochemical site of the herbicide's activity. Its expression is directed to the chloroplasts, the site of the herbicide activity, by the addition of genetic coding sequences of a plant derived transit peptide. No antibiotic resistance marker genes were introduced into GTS 40-3-2.

The GTS 40-3-2 line has been field tested under confined conditions since 1992 in Ontario. It has received complete approval for commercialization in the US.

Monsanto has submitted information and data to CFIA on the identity of GTS 40-3-2, a detailed description of the modification method, the role of the gene and regulatory sequences in the donor organisms, the molecular characterization of the inserted DNA and the insertion site, the stability of the gene insertion, and full nucleotide sequences.

The novel protein was identified and characterized, including its potential toxicity to livestock and non-target organisms, potential for allergenicity, functional activity, expression levels in leaves and seeds, equivalence to the source protein expressed in bacteria, and full amino acid sequences. A number of detailed scientific publications providing further data was also supplied.

Agronomic characteristics such as germination of seeds, final stands, and disease and insect susceptibility, were compared to those of unmodified G. max counterparts. Seed yields of line GTS 40-3-2 and its unmodified counterpart were also compared.

Compositional analyses included measurements of seed amino acids (including aromatic amino acids) and fatty acids, seed protein profiles, ash, gross energy and crude fibre content. The nutrient digestibility for lactating cows was studied, and feeding trials with raw soybeans to dairy cows, rats, and quails, and with soybean meal to broilers, rats, and catfish, were performed. Antinutrients such as trypsin inhibitor, lectins, urease and isoflavones, as well as endogenous allergens, were analyzed in raw seeds.

Monsanto has also supplied a referenced description of the biology of G. max L., with consideration of soybean genetics, taxonomy, life cycle, potential for outcrossing to other soybean plants and to related species, and distribution of these related species in Canada.

The Plant Biosafety Office of the Plant Health and Production Division, has reviewed the above information, in light of the assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08:

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

The Feed Section of the Plant Health and Production Division, CFIA, has also reviewed the above information in light of the assessment criteria for determining safety and efficacy of livestock feed, as described in Dir95-03:

• potential impact on livestock, and
• potential impact on livestock nutrition.

III. Description of the Novel Trait

1. Glyphosate Tolerance

• Glyphosate, the active ingredient of Roundup®, acts as a competitive inhibitor of 5-enol-pyruvylshikimate-3-phosphate synthase (EPSP synthase), an essential enzyme of the shikimate biochemical pathway, involved in the production of the aromatic amino acids phenylalanine, tyrosine and tryptophan. This inhibition results in growth suppression and plant death.
• The inserted glyphosate tolerance gene codes for a bacterial version of this essential enzyme, ubiquitous in plants, fungi and microorganisms. This enzyme is highly insensitive to inhibition by glyphosate and therefore fulfills the aromatic amino acid metabolic needs of the plant.
• A plant-derived DNA sequence coding for a chloroplast transit peptide was co-introduced with the glyphosate tolerance gene. This peptide facilitates the import of the newly translated EPSP synthase into chloroplasts, where both the shikimate pathway and glyphosate site of action are located. The transit peptide is degraded rapidly and completely by proteases after the import has occurred.
• The gene, associated with its transit peptide coding sequence, is linked to a strong constitutive promoter. Protein expression was quantified, and averaged 0.239 µg/mg (f.w.) in seeds and 0.495 µg/mg (f.w.) in leaves.
• The full nucleotide sequence of the inserted gene was provided. The resulting enzyme was shown to be functionally and structurally similar to bacterial enzymes of the same type frequently found in food and feed products. Molecular weights were similar, indicating that the protein had not been glycosylated, nor had it undergone post-transcriptional modifications.
• Studies showed that the introduced EPSP synthase was not heat stable, and that activity was lost after incubation at 65°C for 15 minutes. It was not stable in the presence of proteases in mammalian digestive tract, and was inactivated during processing required to manufacture soybeans into feed ingredients.
• EPSP synthase is ubiquitous in nature and is not expected to be toxic or allergenic to non-target organisms. When subjected to comparative analyses with three genetic sequence databases of toxic or allergenic polypeptides, the enzyme amino acid sequence showed no significant homology with any known toxin or allergen.

2. Development Method

• The soybean commercial variety A5403 (Asgrow Seed Co.) was transformed using particle bombardment, with a modified E. coli plasmid vector containing the genes coding for glyphosate tolerance and for production of ß-glucuronidase, a screenable marker. The original transformant selected showed two insertion sites, one with the screenable marker, and the other with the glyphosate tolerance gene. These two sites subsequently segregated independently in the following sexual generation, and line GTS 40-3-2, upon analysis, was found to contain just one insertion site in which is integrated the glyphosate tolerance gene only.

3. Stable Integration into the Plant's Genome

• The data provided showed that there was no incorporation of any coding region from outside the fusion gene in the original plasmid vector, consisting of the glyphosate tolerance gene, combined with the transit peptide coding sequence, in addition to their regulatory sequences. Only one copy was integrated at the single insertion site.
• Following generations demonstrated no further segregation of the fusion gene described above, showing that line GTS 40-3-2 was homozygous for that fusion gene. DNA analyses over six generations showed that the insertion was stable.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT to Become a Weed of Agriculture or be Invasive of Natural Habitats

CFIA has evaluated data submitted by Monsanto on seed yield, and visual observations on germination characteristics of seeds, final stands, disease and insect susceptibility of GTS 40-3-2, and has determined that it is substantially equivalent to unmodified varieties for these characteristics. No competitive advantage was conferred by the insertion of the novel gene, other than tolerance to the Roundup® herbicide.

Based on the submitted information, CFIA has determined that GTS 40-3-2 does not show any stress adaptation other than its resistance to glyphosate. The line was tested under various environmental conditions, and showed no obvious differences in agronomic performance when compared to unmodified counterparts under the same conditions.

The description of the biology of G. max demonstrates that unmodified plants of this species are not weedy, nor invasive of unmanaged habitats in Canada, and that they are not wind-pollinated and mostly self-pollinated. According to the information provided by Monsanto, GTS 40-3-2 was determined not to be different from its counterparts in this respect. Glyphosate is commonly used for chemical fallow production, nonetheless the emergence of resistant soybean volunteer plants should not be problematic, since soybean does not survive under cool wet soil conditions. Glyphosate resistant volunteer plants, should they arise, can easily be managed by mechanical means and other available chemicals currently used to control soybeans. Glyphosate is not used in normal crop rotation cycles, and resistance is therefore not an issue of concern in weed management control.

The above considerations, together with the fact that the novel trait has no intended effect on invasiveness, have led CFIA to conclude that line GTS 40-3-2, and its progeny have no altered weediness or invasiveness potential compared to currently commercialized soybean varieties.

Note: A longer term concern, if there is general adoption of several different crop and specific herbicide weed management systems, is the potential development of crop volunteers with a combination of novel resistances to different herbicides. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, agricultural extension personnel, in both the private and public sectors, should promote careful management practices for growers who use these herbicide tolerant crops to minimize the development of multiple resistance.

2. Potential for Gene Flow to Wild Relatives whose Hybrid Offspring may Become more Weedy or more Invasive

The description of the biology of G. max shows that natural hybridization between cultivated soybeans and the wild annual species G. soja can occur. Glycine soja is endemic in China, Korea, Japan, Taiwan and the former USSR. It 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.

CFIA therefore concludes that, in Canada, the potential for transfer of the glyphosate tolerance trait from the transgenic line to soybean relatives through gene flow is negligible in managed ecosystems, and that there is no potential for transfer to wild species.

3. Altered Plant Pest Potential

Soybean is not a plant pest in Canada, and the intended effect of the novel trait is unrelated to plant pest potential. In addition, agronomic characteristics, qualitative and quantitative composition of GTS 40-3-2 were shown to be within the range of values displayed by currently commercialized soybean varieties. CFIA has therefore concluded that plant pest potential of this line is not altered.

4. Potential Impact on Non-Target Organisms

The detailed characterization of the novel gene and resulting enzyme, as briefly summarized in Part III of the present document, has led to the conclusion that these do not result in altered toxic or allergenic properties. The enzyme is ubiquitous in microorganisms, fungi and plants, and is rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis.

Raw seeds of 40-3-2 were shown to be substantially equivalent to traditional varieties in terms of antinutritional factors such as trypsin inhibitor, lectins, isoflavones and urease. Seed protein profiles, amino acid and fatty acid compositions were equivalent to those of the unmodified counterpart.

Based on the above, CFIA has determined that, when compared with currently commercialized counterparts, the unconfined release of the GTS 40-3-2 line will not result in altered impacts on interacting organisms, including humans.

5. Potential Impact on Biodiversity

The introduced EPSP synthase was determined to be safe to non-target organisms. In addition, the transgenic line has no novel phenotypic characteristic which would extend its use beyond the current geographic range of soybean production in Canada. Since there are no wild relatives of soybeans in Canada, and since soybean is not an invasive species, the novel trait will not be transferred to unmanaged environments.

CFIA has therefore concluded that the potential impact on biodiversity of the GTS 40-3-2 line is equivalent to that of currently commercialized soybean lines.

V. Assessment Criteria for Use as Livestock Feed

1. Anti-Nutritional Factors

95% Confidence Intervals were determined for trypsin inhibitor activity and urease content of the raw seeds from GTS 40-3-2 and the control variety A5403 grown under a variety of conditions. No significant differences were observed.

2. Nutritional Composition of PNT

No statistical differences in nutritional composition, i.e., ash, gross energy, and crude fibre content, were noted between the raw whole seed and the control variety A5403. The crude protein and crude fat content of raw seeds was not significantly different from that of the control line in two of three studies. The nutrient digestibility of GTS 40-3-2 was found to be substantially equivalent to traditional varieties per a 29-day lactation trial wherein lactating cows were fed 10% raw soybeans. The levels of the amino acids phenylalanine, tryptophan, and tyrosine were found to be substantially equivalent to those of traditional varieties. These results collectively demonstrate that the introduction of DNA into G. max resulting in GTS 40-3-2, did not likely result in any secondary effects impacting on the composition or nutritional quality of the cultivar. Accordingly, GTS 40-3-2 was judged to be substantially equivalent to traditional soybean varieties in terms of nutritional composition.

3. Feeding Trials

Soybean from GTS 40-3-2 and the control variety A5403 were fed to dairy (raw soybeans) for 29 days, and to broilers (soybean meal) for 6 weeks. Livestock from both studies exhibited equivalent performance.

VI. Regulatory Decision

Based on the review of data and information submitted by Monsanto, and through comparisons of the transgenic lines with unmodified soybean counterparts, CFIA has concluded that neither the novel gene nor its resulting gene product and associated novel trait confer any intended or unintended ecological advantage, or environmental impact, to GTS 40-3-2. The potential for transfer to other soybean plants is very low, due to the biology of the species. There is no potential for transfer to wild relatives.

In addition, CFIA has concluded that the novel gene and its corresponding trait do not in themselves raise any concerns regarding the safety or nutritional composition of this line as feed. Soybean meal and flakes are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As GTS 40-3-2 has been assessed and found to be substantially equivalent to traditional soybean varieties, GTS 40-3-2 and its byproducts are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada.

Unconfined release into the environment, including feed use of line GTS 40-3-2, and other G. max lines derived from it, but without the introduction of any other novel traits, is therefore considered safe.

This bulletin is published by the Plant Health and Production Division. For further information, please contact the Plant Biosafety Office or the Feed Section at: