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Canadian Food Inspection Agency - Plant Biosafety Office - Supplement to Decision Doc. Page 1 of 6 Plants > Biotechnology / PNTs > Unconfined Release > Decisions Supplement to Decision Document DD1999-30:
Determination of Livestock Feed Safety of Aventis CropScience
Canada Inc.'s Glufosinate Ammonium Tolerant Soybean
(Glycine max) Events A2704-12 and A5547-127
Decision Document 99-30
This Decision Document has been prepared to explain the regulatory decision reached under regulatory directive Dir95-03 Guidelines for the Assessment of Novel Feeds: Plant Sources. The Canadian Food Inspection Agency (CFIA), specifically the Feed Section of the Animal Health and Production Division, has evaluated information submitted by Aventis CropScience Canada Inc. regarding soybean events A2704-12 and A5547-127. This information is in regard to the tolerance of soybean events A2704-12 and A5547-127 to glufosinate ammonium herbicide Liberty™. The CFIA has determined that these events do not present livestock feed safety concerns when compared to currently commercialized soybean varieties in Canada. Taking into account this evaluation, livestock feed use of soybean events A2704-12 and
A5547-127 is therefore authorized by the Feed Section of the Animal Health and
Production Division as of December 18, 2000. Any soybean lines derived from events
A2704-12 and A5547-127 may also be used as livestock feed, 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
livestock feed safety and (iv) the novel genes are expressed at a level similar to that of
the authorized line.
Soybean events A2704-12 and A5547-127 are subject to the same phytosanitary import
requirements as their unmodified counterparts.
Please note that an assessment of livestock feed safety is a critical step in the potential commercialization of these plant types. Other requirements, such as an evaluation of food safety by Health Canada, have been addressed separately from this review. Table of Contents
I. Brief Identification of the Modified Plant 2. Glufosinate Ammonium Tolerance 3. Ampicillin Resistance IV. Criteria for the Livestock Feed Assessment 1. Potential Impact of Soybean Events A2704-12 and A5547-127 on Livestock Nutrition 2. Potential Impact of Soybean Events A2704-12 and A5547-127 on Livestock and http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9930supe.shtml Canadian Food Inspection Agency - Plant Biosafety Office - Supplement to Decision Doc. Page 2 of 6 I. Brief Identification of the Modified Plant
Designation(s) of the Modified Plant: Soybean events A2704-12 and A5547-127, OECD Unique
Identifier ACS-GM005-3 and ACS-GM006-4
Applicant: Aventis CropScience Canada Inc.
Plant Species: Soybean (Glycine max (L.) Merr.)
Novel Trait: Tolerance to glufosinate ammonium herbicide, the active ingredient of Liberty™
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 II. Background Information
Aventis CropScience Canada Inc. has developed two soybean events, A2704-12 and A5547-127, that are tolerant to Liberty™, a broad spectrum, non-residual, glufosinate ammonium herbicide. These soybean events 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 events A2704-12 and A5547-127 was based on recombinant DNA technology through which the bacterial based synthetic pat gene was introduced into the soybean commercial cultivars "A2704" and "A5547", respectively. 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 or A5547-127 and consequently no gene product from the ampicillin resistance gene was detected. Aventis CropScience Canada Inc. has submitted information and data to the CFIA on the identity of events A2704-12 and A5547-127, the molecular characterization, a detailed description of the modification method, data and information on the stability of the gene insertions, the role of the inserted genes 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 events A2704-12 and A5547-127 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. Nutritional components of the soybean events A2704-12 and A5547-127 such as proximates, amino acids and fatty acids were compared with unmodified soybean counterparts. Anti-nutritional http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9930supe.shtml Canadian Food Inspection Agency - Plant Biosafety Office - Supplement to Decision Doc. Page 3 of 6 The Feed Section, CFIA, has 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 Feed Section has considered: z potential impact of soybean events A2704-12 and A5547-127 on livestock nutrition; and z potential impact of soybean events A2704-12 and A5547-127 on livestock and workers/by- III. Description and Assessment of the Novel Trait
1. Development Method
The soybean commercial cultivars "A2704" and "A5547" (Asgrow Seed Co.) were transformed using particle bombardment with a modified E. coli plasmid vector containing the gene coding for 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 The pat gene introduced into Aventis CropScience Canada Inc.'s soybean events A2704-12 and A5547-127 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. 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 events A2704-12 and A5547-127 is unlikely to have been glycosylated or undergone other post translational modifications. Proteins produced in procaryotes (bacterial systems) are generally not subject to post translational modifications. The molecular weight of the PAT enzyme from the plant derived sources was similar for soybean events A2704-12 and A5547-127 and corn lines T14 and T25 (see 3. Ampicillin Resistance
An ampicillin resistance gene was used as a selectable marker during the development of the genetic sequence inserted into soybean events A2704-12 and A5547-127. This gene was not intended to have an agronomic or livestock feed purpose. http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9930supe.shtml Canadian Food Inspection Agency - Plant Biosafety Office - Supplement to Decision Doc. Page 4 of 6 Analysis of events A2704-12 and A5547-127 demonstrated that they did not possess an intact sequence of the ampicillin resistance gene. Furthermore, the ampicillin resistance gene introduced into these events 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 and one copy of the pat gene in event A5547-127. DNA analyses carried out on plants from both events 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 IV. Criteria for the Livestock Feed Assessment
1. Potential Impact of Soybean Events A2704-12 and A5547-127 on
Livestock Nutrition
Nutritional composition
Crude protein, fat, fibre, ash, amino acids, fatty acids, calcium and phosphorous were determined for soybean events A2704-12 and A5547-127 and their non-transgenic counterparts, at nine locations in Canada and the United States. For some of these parameters, occasional small but significant differences were observed. These differences were not consistent across locations. Additionally, the composition of the transgenic and control plants was within the expected range for soybean. Antinutritional factors
Trypsin inhibitor activity and isoflavone levels were determined for soybean events A2704-12 and A5547-127 and their non-transgenic counterparts at nine locations in Canada and the United States. The analyses were done on seed, forage and hay. Trypsin inhibitor activity in seeds from A5547-127 was higher than its respective control. Trypsin inhibitor activity in a seed sample from soybean event A2704-12 was higher than its respective control at one of the U.S. sites. However, these differences were not considered to be of nutritional significance. Feeding Trials
Soybeans from events A2704-12 and A5547-127, and appropriate controls, were fed to broilers at 20% of the ration for 15 and 42 days, respectively. Based on the parameters observed (feed intake, feed efficiency, body weight, mortality, carcass characteristics), it can be concluded that soybean events A2704-12 and A5547-127 are substantially equivalent, in terms of feed performance, to their non-transgenic counterpart. Conclusion
The evidence provided by Aventis CropScience Canada Inc. supports the conclusion that the nutritional composition of soybean events A2704-12 and A5547-127 is substantially equivalent to 2. Potential Impact of Soybean Events A2704-12 and A5547-127 on
Livestock and Workers/By-standers
http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9930supe.shtml Canadian Food Inspection Agency - Plant Biosafety Office - Supplement to Decision Doc. Page 5 of 6 Soybean events A2704-12 and A5547-127 were tested for allergenic potential through the use of RAST (RadioAllergoSorbent Test) based assays and IgE based immunoblot assays. The results of these tests indicated that soybean events A2704-12 and A5547-127 do not have increased allergenicity potential in comparison to an unmodified counterpart. PAT protein analyses used an E. coli-produced PAT protein that was shown to be equivalent to that present in events A2704-12 and A5547-127. The PAT protein was shown to degrade rapidly in various gastric fluids and to lack any nucleotide or amino acid sequence similarity with any known toxins or allergens. Additionally, acute oral gavage rat and intravenous injection mice studies demonstrated a lack of toxic effects for the PAT protein. Based on the information provided by Aventis CropScience Canada Inc., the PAT protein is unlikely Based on the detailed characterization provided (nutritional composition and agronomic data of the modified plants compared to unmodified comparators) it is unlikely that any unintended effects are present in soybean events A2704-12 and A5547-127. The evidence provided by Aventis CropScience Canada Inc. supports the conclusion that the potential impact on livestock and workers/by-standers of soybean events A2704-12 and A5547-127 is equivalent to that of currently commercialized soybean lines. V. New Information Requirements
If at any time, Aventis CropScience 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 events A2704-12 and A5547-127 material in Canada or elsewhere, Aventis CropScience 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 events A2704-12 and A5547-127 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use authorization of soybean events A2704-12 and A5547-127. VI. Regulatory Decision
Based on the review of data and information submitted by Aventis CropScience Canada Inc., including comparisons of soybean events A2704-12 and A5547-127 with unmodified soybean counterparts, the Feed Section of the Animal Health and Production Division, has concluded that the introduced genes and their corresponding novel trait will not confer to these plants any characteristics that would raise any concerns regarding the safety or nutritional composition of soybean events A2704-12 and A5547-127. 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 events A2704-12 and A5547-127 have been assessed and found to be as safe as nutritious as traditional soybean varieties. Soybean events A2704-12 and A5547-127 and their products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. Taking into account this evaluation, livestock feed use of soybean events A2704-12 and
A5547-127 is therefore authorized by the Feed Section of the Animal Health and
Production Division as of December 18, 2000. Any soybean lines derived from these
soybean events may be imported and/or released, provided no inter-specific crosses are
performed, provided the intended uses are similar, 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 specific
use and safety for the environment and for human and animal health and the novel
genes are expressed at a level similar to that of the authorized line.
http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9930supe.shtml Canadian Food Inspection Agency - Plant Biosafety Office - Supplement to Decision Doc. Page 6 of 6 Soybean events A2704-12 and A5547-127 are subject to the same phytosanitary import
requirements as their unmodified counterparts.
Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of soybean events A2704-12 and A5547-127. The food safety decisions are available at the following Health Canada web site: http://www.hc-sc.gc.ca/fn-an/gmf-agm/appro/index-eng.php 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: Plant Products Directorate 59 Camelot Drive, Nepean Ontario, K1A 0Y9
613-225-2342
59 Camelot Drive, Nepean
Ontario, K1A 0Y9
613-225-2342
http://www.inspection.gc.ca/english/plaveg/bio/dd/dd9930supe.shtml

Source: http://cera-gmc.org/docs/decdocs/09-052-006.pdf