DD1999-30: Determination of Environmental Safety of AgrEvo Canada Inc.'s Glufosinate Ammonium Tolerant Soybean (Glycine max) Event A2704-12

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Supplement to Decision Document DD99-30

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" and its companion biology document BIO1996-10, "The Biology of Glycine max (L.) Merr. (Soybean)".

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Products Directorate, has evaluated information submitted by AgrEvo Canada Inc. regarding soybean event A2704-12. This information is in regard to the tolerance of soybean event A2704-12 to glufosinate ammonium herbicide Liberty™. The CFIA has determined that this plant with a novel trait does not present a greater risk to the environment when compared to currently commercialized soybean varieties in Canada.

Taking into account this evaluation, unconfined release into the environment of soybean event A2704-12 is therefore authorized by the Plant Biosafety Office of the Plant Products Directorate April 26, 1999. Any soybean lines derived from event A2704-12 may also be released into the environment provided (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 soybean varieties in Canada, in terms of their potential environmental impact and (iv) the novel gene is expressed at a level similar to that of the authorized line.

Soybean event A2704-12 is subject to the same phytosanitary import requirements as its unmodified counterparts.

Please note that the assessment of environmental safety of PNTs is a critical step 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

  1. Brief Identification of the Modified Plant
  2. Background Information
  3. Description of the Novel Trait
    1. Development Method
    2. Glufosinate Ammonium Tolerance
    3. Ampicillin Resistance
    4. Stable Expression
  4. Assessment Criteria for Environmental Safety
    1. Potential of Soybean Event A2704-12 to Become a Weed of Agriculture or Invasive of Natural Habitats
    2. Potential for Gene Flow from Soybean Event A2704-12 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Plant Pest Potential of Soybean Event A2704-12
    4. Potential Impact on Non-Target Organisms of Soybean Event A2704-12
    5. Potential Impact on Biodiversity of Soybean Event A2704-12
  5. New Information Requirements
  6. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation(s) of the Modified Plant: Soybean event A2704-12, OECD Unique Identifier ACS-GM005-3

Applicant: AgrEvo Canada Inc.

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

Novel Traits: Tolerance to glufosinate ammonium herbicide, the active ingredient of Liberty™ herbicide

Trait Introduction Method: Particle bombardment (biolistics)

Proposed Use of the Modified Plant: Commercial production of soybean varieties in Canada for animal feed (mostly defatted toasted meal and flakes) and human consumption (mostly oil, protein fractions and dietary fibre). These plants are not intended to be grown outside the normal cultivation area for soybean in Canada.

II. Background Information

AgrEvo Canada Inc. has developed a soybean event, designated A2704-12, that is tolerant to Liberty™, a broad spectrum, non-residual, glufosinate ammonium herbicide. This soybean event will allow the use of Liberty™ as a post-emergence herbicide, thus providing an alternative for weed control in soybean production, which may reduce reliance on soil-incorporated herbicides.

The development of soybean event A2704-12 was based on recombinant DNA technology through which the bacterial based synthetic pat gene was introduced into the soybean commercial cultivar "A2704". This gene codes for phosphinothricin acetyltransferase (PAT), an enzyme that inactivates glufosinate ammonium through acetylation. The bla gene, conferring resistance to ampicillin antibiotic, was included in the original genetic sequence, but no intact copies were detected in event A2704-12 and consequently no gene product from the ampicillin resistance gene was detected. The ampicillin gene is of no agronomic interest and was used as a selectable marker in the development of the genetic sequence carrying the herbicide tolerance gene.

Soybean event A2704-12 was field tested in Ontario, Canada under confined conditions in 1997 and 1998.

AgrEvo Canada Inc. has submitted information and data to the CFIA on the identity of event A2704-12, the molecular characterization, a detailed description of the modification method, data and information on the stability of the gene insertion, the role of the inserted gene and regulatory sequences in donor organisms. The novel protein was identified and information on its expression in seeds, plant tissue, forage and hay was provided. The novel gene product present in soybean event A2704-12 is identical to the gene product approved by the CFIA for canola and corn as described in the following decision documents: DD95-01 AgrEvo Canada Inc.'s Glufosinate Ammonium-Tolerant Canola, DD96-11 AgrEvo Canada Inc.'s Glufosinate Ammonium-Tolerant Canola line HCN28, and DD98-22 Determination of the Safety of AgrEvo Canada Inc.'s Glufosinate Ammonium Tolerant Corn (Zea mays) lines, T14 and T25.

Agronomic characteristics such as maturity, yield, plant height, plant lodging, oil and protein content, and disease and insect susceptibility were compared to those of its unmodified soybean counterpart.

The Plant Biosafety Office, CFIA, has reviewed the above information, with respect to the assessment criteria for determining environmental safety of plants with novel traits, as described in Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". The Plant Biosafety Office has considered:

  • potential of soybean event A2704-12 to become a weed of agriculture or be invasive of natural habitats;
  • potential for gene-flow from soybean event A2704-12 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • potential of soybean event A2704-12 to become a plant pest;
  • potential impact of soybean event A2704-12 or its gene products on non-target species, including humans; and
  • potential impact of soybean event A2704-12 on biodiversity.

III. Description and Assessment of the Novel Trait

1. Development Method

The soybean commercial cultivar "A2704" (Asgrow Seed Co.) was transformed using particle bombardment with a modified E. coli plasmid vector containing the gene coding for tolerance to glufosinate ammonium. The plant cells were regenerated and transformants were selected by spraying plantlets in aseptic conditions with glufosinate ammonium.

Homozygous plants were selected from those in which all progeny were tolerant to glufosinate ammonium.

2. Glufosinate Ammonium Tolerance

Phosphinothricin, the active ingredient of glufosinate ammonium, inhibits the enzyme glutamine synthetase, which results in the accumulation of lethal levels of ammonium in susceptible plants within hours of application.

The pat gene introduced into AgrEvo Canada Inc.'s soybean event A2704-12 codes for phosphinothricin-acetyltransferase (PAT). This enzyme detoxifies L-phosphinothricin by acetylation into an inactive compound. PAT has extremely high substrate specificity for L-phosphinothricin, the active component of the herbicide Liberty™. Studies on the specificity of the acetyltransferase clearly demonstrated that neither L-phosphinothricin's analog L-glutamic acid nor any other amino acid can be acetylated by the PAT enzyme.

The synthetic PAT gene was derived from a PAT gene isolated from Streptomyces viridochromogenes, an aerobic soil bacteria. The PAT enzyme occurs naturally in the soil and acetyltransferases are ubiquitous in nature.

The PAT gene is linked to a constitutive promoter, and protein expression was detected in seed, plant tissues, forage and hay.

Proteins produced in procaryotes (bacterial systems) are generally not subject to post translational modifications. Consequemtly, similar molecular weights found between the PAT enzyme derived from the modified plants and from a bacterial expression system indicates that the protein in soybean event A2704-12 is unlikely to have been glycosylated or undergone other post translational modifications. The molecular weight of the PAT enzyme from soybean event A2704-12 is similar to the molecular weight of the PAT enzymes from corn lines T14 and T25 (see DD98-22).

3. Ampicillin Resistance

An ampicillin resistance gene was used as a selectable marker during the development of the genetic sequence inserted into soybean event A2704-12. This gene was not intended to have an agronomic purpose.

Analysis of event A2704-12 demonstrated that it did not possess an intact sequence of the ampicillin resistance gene. Furthermore, the ampicillin resistance gene introduced into this event is not functional as it does not have the necessary regulatory sequences for expression in plants.

4. Stable Expression

The data provided demonstrated the stable integration of two copies of the pat gene in transformation event A2704-12. DNA analyses carried out on plants from this event over three generations (R3, R4, and R5) demonstrated that the insertions were stable.

Data provided on segregation ratios demonstrated that the pat gene was dominant and inherited in a Mendelian fashion in the event.

IV. Assessment Criteria for Environmental Safety

1. Potential of Soybean Event A2704-12 to Become a Weed of Agriculture or 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 weedy, nor invasive of unmanaged habitats in Canada, and that they are not wind-pollinated and mostly self-pollinated. Soybean event A2704-12 was determined not to be different from its traditionally developed counterparts in this respect.

According to the information provided by AgrEvo Canada Inc., no competitive advantage was conferred to soybean event A2704-12, other than that conferred by tolerance to the Liberty™ herbicide. The CFIA has evaluated data submitted by AgrEvo Canada Inc. on the reproductive and survival biology of event A2704-12 and determined that morphological traits, plant maturity, yield, disease and insect resistance, and quality traits were within the normal range of expression of characteristics in unmodified soybean counterparts. Soybean event A2704-12 did not show any stress adaptation other than its tolerance to glufosinate ammonium herbicide. It is therefore not expected that soybean event A2704-12 would possess traits that would render it invasive of natural habitats since none of the reproductive or growth characteristics were modified.

Glufosinate ammonium tolerance in itself will not cause soybean event A2704-12 to become more weedy in managed habitats than non-transformed soybean. Glufosinate ammonium tolerant soybean volunteer plants can easily be managed by mechanical means or by the use of other available herbicides.

The above considerations have led the CFIA to conclude that the soybean event A2704-12 has no altered weed or invasiveness potential compared to currently commercialized soybean varieties.

Note: If there is general adoption of different herbicide weed management systems, then the potential exists for the development of crop volunteers with a combination of novel tolerances to different herbicides. As a result, this technology should be managed as part of an integrated approach which may include currently available weed control products with alternate modes of action, or alternative methods of weed control. Of additional note is the use several crop species in rotation which all rely on tolerance to the same herbicide. The continued use of a specific herbicide may provide significant selective pressure for the potential development of herbicide resistant weeds. 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 herbicide tolerant crop volunteers as well as tolerant weed populations.

2. Potential for Gene Flow from Soybean Event A2704-12 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of soybean, as described in BIO1996-10, shows that natural hybridization between cultivated soybean and the wild annual species Glycine 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.

The CFIA has therefore concluded that the potential for transfer of the glufosinate ammonium tolerance trait from soybean event A2704-12 to soybean relatives through gene flow is negligible in managed or unmanaged ecosystems

3. Altered Plant Pest Potential of Soybean Event A2704-12

Soybean is not a plant pest in Canada and the novel trait in soybean event A2704-12 is not expected to affect its plant pest potential.

The intended effect of the novel trait is unrelated to plant pest potential. Morphological and agronomic characteristics of soybean event A2704-12 were shown to be within the range of values displayed by currently commercialized soybean varieties, leading to the conclusion that plant pest potential was not inadvertently altered.

The CFIA have therefore determined that soybean event A2704-12 does not present a plant pest concern.

4. Potential Impact on Non-Target Organisms of Soybean Event A2704-12

The PAT enzyme responsible for glufosinate ammonium tolerance has very specific enzymatic activity and does not affect the metabolism of the plant. In addition, the PAT enzyme does not possess proteolytic or heat stability, therefore it will not be expected to be stable in the environment. PAT is rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis. PAT does not contain potential glycosylation sites nor does it possess proteolytic or heat stability, indicating that PAT is not a likely allergen. A search of the GENEBANK DNA sequence database revealed no significant homology with any known toxins or allergens.

Other crops have been modified by recombinant DNA techniques to express the PAT enzyme with no apparent effect on the agronomic performance of succeeding crops. The crops being tested included wheat, barley, lentils, peas, flax and alfalfa and are not all approved in Canada. Expression levels of PAT in soybean event A2704-12 are comparable to other transformed crop species. Therefore, the CFIA concludes that no residual effects are expected on crops following soybean event A2704-12 production.

Based on the above, the CFIA has determined that the unconfined release of soybean event A2704-12 will not result in altered impacts on non-target organisms, including humans, when compared with currently commercialized counterparts.

5. Potential Impact on Biodiversity of Soybean Event A2704-12

Soybean event A2704-12 has no novel phenotypic characteristics which would extend its use beyond the current geographic range of soybean production in Canada. In addition, soybean event A2704-12 was shown to be safe to non-target organisms and does not present altered weediness or plant pest potential. Since soybean has no wild relatives in Canada that it can outcross to, there will be no transfer of novel traits to unmanaged environments.

The CFIA has therefore concluded that the impact on biodiversity of soybean event A2704-12 is equivalent to that of currently commercialized soybean lines.

V. New Information Requirements

If at any time, AgrEvo 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 A2704-12 material in Canada or elsewhere, AgrEvo 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 soybean event A2704-12 on the environment, livestock and human health, and may re-evaluate its decision with respect to the environmental release authorization of soybean event A2704-12.

VI. Regulatory Decision

Based on the review of data and information submitted by AgrEvo Canada Inc., and through comparisons of soybean event A2704-12 with unmodified soybean counterparts, the Plant Biosafety Office, CFIA, has concluded that the modified gene and its corresponding novel trait will not confer to soybean event A2704-12 any characteristic that would result in unintended environmental effects following unconfined release.

Taking into account this evaluation, unconfined release into the environment of soybean event A2704-12 is therefore authorized by the Plant Biosafety Office of the Plant Products Directorate as of April 26, 1999. Any soybean lines derived from soybean event A2704-12 may also be released into the environment, provided no interspecific 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 soybean varieties in Canada, in terms of their potential environmental impact and the novel gene is expressed at a level similar to that of the authorized line.

Soybean event A2704-12 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 A2704-12.

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