DD2005-53: Determination of the Safety of Monsanto Canada Inc.'s Roundup Ready® Alfalfa (Medicago sativa L.) Events J101 and J163

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Issued: 2005-07

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 Bio2005-02, The Biology of Medicago sativa L. (Alfalfa), 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 (PBO) of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate has evaluated information submitted by Monsanto Canada Inc. This information is in regard to the glyphosate tolerant alfalfa events J101 and J163. The CFIA has determined that these plants with a novel trait (PNT) do not present altered environmental risk nor, as a novel feed, do they present livestock feed safety concerns when compared to currently commercialized alfalfa varieties in Canada.

Unconfined release into the environment and use as livestock feed of the alfalfa events J101 and J163 are therefore authorized as of July 28, 2005. Any other alfalfa lines and intraspecific hybrids resulting from the same transformation events and all their descendants, may also be released into the environment and used as livestock feed, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown alfalfa, in terms of their potential environmental impact and livestock feed safety.

The J101 and J163 alfalfa events are subject to the same phytosanitary import requirements as their unmodified counterparts.

Please note, that the livestock feed and environmental safety of PNTs and novel feeds 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 Plants with a Novel Trait (PNT)

II. Background Information

III. Description of the Novel Trait

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

IV. Assessment Criteria for Environmental Safety

  1. Potential of these PNTs 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

V. Criteria for Livestock Feed Assessment

  1. Potential Impact on Livestock Nutrition
  2. Potential Impact on Livestock and Workers/By-Standers

VI. New Information Requirements

VII. Regulatory Decision

I. Brief Identification of the Plants with a Novel Trait (PNT)

Designation(s) of the PNT: Event J101 and J163, OECD identifier MON-00101-8 (event J101), OECD identifier MON-00163-7 (event J163)

Applicant: Monsanto Canada, Inc.

Plant Species: Medicago sativa L.

Novel Trait(s): Herbicide tolerance (glyphosate).

Trait Introduction Method: Agrobacterium-mediated transformation

Proposed Use of PNTs: Production of M. sativa for seed and forage for livestock feed.

II. Background Information

Monsanto Canada Inc., in collaboration with Forage Genetics International (FGI), has developed Roundup Ready® Alfalfa events J101 and J163, containing an epsps gene, coding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This gene imparts tolerance to glyphosate (the active ingredient in Roundup® herbicide), a novel trait in alfalfa varieties currently cultivated in Canada. This herbicide tolerance trait allows for the control or suppression of economically important weeds in alfalfa production.

The development of the glyphosate tolerant alfalfa events was accomplished with recombinant DNA technology. The epsps coding sequence from the bacterium Agrobacterium sp. strain CP4 (i.e cp4 epsps), was introduced into alfalfa clone R2336. Line R2336 was selected from an elite, high yielding, fall-dormant FGI alfalfa breeding population using a tissue culture screen for callus formation and somatic embryo induction. Resultant calluses which produced the CP4 EPSPS protein were identified by tolerance to exposure to the herbicide glyphosate. The CP4 EPSPS protein imparts reduced sensitivity, in the chloroplast, to glyphosate herbicides. The cp4 EPSPS coding sequence in alfalfa events J101 and J163 is fused to a chloroplast transit peptide (CTP) sequence which directs the translated protein to the chloroplast, the site of amino acid biosynthesis.

If commercialized, Roundup Ready® alfalfa varieties will use two different cp4 epsps insertion events (J101 and J163) combined through a conventional breeding process.

Monsanto Canada Inc. has provided data on the identity of events J101 and J163, a detailed description of the transformation methods, data and information on the gene insertion sites, copy numbers and levels of expression in the plants, the role of the inserted genes and regulatory sequences in donor organisms and the amino acid sequences. Due to the low levels of CP4 EPSPS protein expressed in these alfalfa plants it was necessary to produce CP4 EPSPS protein in a microbial expression system to obtain sufficient quantities of the protein in order to conduct some of the protein characterization studies. References to relevant scientific publications were included.

These plants have been field tested in Canada under confined conditions in Manitoba and Ontario in 2002 and 2001 respectively. Field trials had also been conducted in the USA, between 1998 and 2003.

Agronomic characteristics of Roundup Ready® alfalfa populations containing events J101 and J163, such as forage and seed yield, vegetative vigour, stand longevity and changes in susceptibilities to various pests and diseases, were compared to those of unmodified M. sativa counterparts.

Monsanto Canada Inc. has provided an agronomic stewardship plan for Roundup Ready® alfalfa in the Canadian environment. This plan includes information regarding a safe and sustainable deployment of Roundup Ready® alfalfa and an efficient mechanism for growers to report agronomic problems with this product to Monsanto Canada Inc.

Nutritional components of Ready alfalfa events J101 and J163 such as proximates, amino acids and fatty acids were compared with unmodified alfalfa counterparts.

The Plant Biosafety Office (PBO) of the Plant Health and Biosecurity Directorate, CFIA, has 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 PBO has considered:

  • potential of the PNTs to become weeds 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 PNTs to become plant pests,
  • potential impact of the PNTs or their gene products on non-target species, including humans, and
  • potential impact on biodiversity.

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

  • potential impact to livestock and workers/by-standers; and,
  • potential impact on livestock nutrition.

III. Description of the Novel Trait

1. Glyphosate Tolerance

  • A gene derived from an Agrobacterium sp. strain CP4 (cp4 epsps) which imparts field level tolerance to glyphosate, the active ingredient in Roundup® agricultural herbicides, was introduced into alfalfa to produce events J101 and J163.
  • An optimized chloroplast transit peptide isolated from Arabidopsis thaliana 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.
  • The inserted gene codes for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimic acid metabolic pathway which is essential for the production of the aromatic amino acids. The native alfalfa EPSPS enzyme is inhibited by glyphosate. The herbicide disrupts the shikimic acid pathway, leading to growth suppression or death of the plant. The bacterial CP4 EPSPS version of this enzyme, expressed in alfalfa events J101 and J163, is not inhibited by glyphosate and continues to catalyze the production of aromatic amino acids in the presence of glyphosate. The expression of the novel enzyme in the plant was quantified by enzyme-linked immunosorbent assay (ELISA).
  • A gene coding for CP4 EPSPS was introduced and expressed in the bacterial species E. coli and the protein was shown to be biologically, chemically and functionally equivalent to the CP4 EPSPS protein produced in the alfalfa plant. Due to the low levels of CP4 EPSPS in the alfalfa plant tissue E. coli produced CP4 EPSPS was used to generate sufficient quantities of pure protein for safety studies. References to relevant scientific publications were included.
  • The CP4 EPSPS protein was found to be labile to mammalian digestion. The data demonstrated a half-life for CP4 EPSPS protein of less than 15 seconds in simulated gastric fluid (pepsin) and within 10 minutes in simulated intestinal fluid. Results were shown on western blots.
  • 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.
  • The amino acid sequence of the CP4 EPSPS protein shares no structurally significant sequence similarity to known toxins and allergens. EPSPS is an enzyme present in many foods with a long history of safe use in Canada, and therefore the CP4 EPSPS form is not be expected to be toxic or allergenic.

2. Development Method

Roundup Ready® alfalfa events J101 and J163 were generated using Agrobacterium-mediated transformations of alfalfa callus (from alfalfa line R2336). A double border, binary vector, designated as PV-MSHT4, containing the epsps gene from the Agrobacterium sp. strain CP4 (CP4 EPSPS) was used. The cp4 epsps expression cassette contains the cp4 epsps coding sequence under the regulation of the figwort mosaic virus (FMV) promoter, a heat shock protein intron (HSP70), a chloroplast transit peptide (CTP2) sequence and a E9 3' polyadenylation sequence. Glyphosate tolerant transformed calluses were selected for elevated levels of glyphosate tolerance through the addition of glyphosate to the plant tissue culture media, then cultured in tissue culture medium for plant regeneration.

3. Stable Integration into the Plant Genome

Both events contain a single copy of the cp4 epsps gene cassette at a single locus of integration. The cp4 epsps gene cassette for both events is intact. No additional elements from the transformation vector PV-MSHT4 were detected in Roundup Ready® events J101 and J163. These data, generated through Southern blot analyses, support the conclusion that only the expected, full length CP4 EPSPS protein is encoded by the inserts in Roundup Ready® events J101 and J163.

Stability analysis for Roundup Ready® alfalfa events J101 and J163 has demonstrated that each event was observed to be stable in the initial generation transformed, subsequent outcrosses and the combination of events J101 and J163 through conventional breeding.

IV. Assessment Criteria for Environmental Safety

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

The biology of alfalfa (Medicago sativa L.) as described in Biology Document Bio2005-02, "The Biology of Medicago sativa L.", shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. There is no evidence in Canada that M. sativa has weed or pest characteristics. According to the information provided by Monsanto Canada, Inc., events J101 and J163 were determined not to be different from their unmodified counterparts in this respect.

The CFIA evaluated data submitted by Monsanto Canada Inc. on the reproductive and survival biology of alfalfa events J101 and J163, and determined that stand establishment, enhanced growth, vigour or stand longevity; changes in susceptibility to plant pests and diseases common to alfalfa; increases in forage and seed yield and increases in seed dormancy were within the normal range of expression of these traits currently displayed by commercial alfalfa varieties.

No competitive advantage was conferred to these plants, other than that conferred by tolerance to glyphosate herbicide. Tolerance to Roundup® agricultural herbicides will not, in itself, render alfalfa weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified.

The above considerations, together with the fact that the novel traits have no intended effects on alfalfa weediness or invasiveness, led the CFIA to conclude that alfalfa events J101 and J163 have no altered weed or invasiveness potential compared to currently commercialized alfalfa.

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 tolerant stewardship plan includes recommendations on agricultural practices concerning Roundup Ready® alfalfa and provides an efficient mechanism for growers to report agronomic problems with this product to Monsanto Canada Inc., facilitating the ongoing monitoring of Roundup Ready® alfalfa.

A longer term consideration, if there is general adoption of several different crop species and specific herbicide weed management systems (ie. numerous combinations of crop species and tolerances to different herbicides), is the potential development of crop volunteers with a combination of novel tolerances to different herbicides. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, Monsanto Canada Inc. will make their stewardship plan readily available to growers and agriculture extension personnel, in both private and public sectors, to promote the careful management practices, such as use of alternate control tools as appropriate to achieve complete control, recommended to help minimize the development of resistant weed populations.

2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of alfalfa, as described in Bio2005-02, indicates that the only wild relative occurring in Canada is M. lupulina. It has been reported that hybrids of M. lupulina and M. sativa are considered impossible, or exceedingly unlikely, to occur in nature.

The CFIA therefore concludes that gene flow from alfalfa events J101 and J163 to wild relatives of alfalfa is highly unlikely in Canada.

3. Altered Plant Pest Potential

The intended effect of the novel trait is unrelated to plant pest potential, and alfalfa is not considered a plant pest in Canada (Bio2005-02). In addition, agronomic characteristics of the modified alfalfa populations were shown to be within the range of values displayed by currently commercialized alfalfa varieties, and indicate that the growing habit of alfalfa was not inadvertently altered. Volunteer alfalfa containing glyphosate tolerance, originating from previous crop years or cross pollination (i.e. wind or bee mediated), can still be managed by growers through the use of alternative herbicides with different modes of action, or cultivation practices which do not involve the use of herbicides. Field observations did not indicate modifications of disease and pest susceptibilities.

The CFIA has therefore determined that the events J101 and J163 do not display any altered pest potential.

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 the expression of the novel protein does not result in altered toxic or allergenic properties. The EPSPS protein is not a known toxin and is commonly found in a wide variety of plants and micro-organisms with a history of safe use. Additionally, acute mouse gavage tests demonstrated the lack of acute oral toxicity of the EPSPS protein.

Based on the above, the CFIA has determined that the unconfined release of alfalfa events J101 and J163 will not result in altered impacts on interacting organisms, including humans, compared to current alfalfa varieties.

5. Potential Impact on Biodiversity

Events J101 and J163 express no novel phenotypic characteristics which could extend its use beyond the current geographic range of alfalfa production in Canada. Since alfalfa does not outcross to wild relatives in Canada, with the possible exception to feral alfalfa populations, there will be no transfer of novel traits to related species in unmanaged environments. In addition the novel trait was determined to pose minimal risks to non-target organisms.

The CFIA has therefore concluded that the potential impact on biodiversity of alfalfa events J101 and J163 is equivalent to that of currently commercialized alfalfa varieties.

V. Criteria for Livestock Feed Assessment

1. Potential Impact on Livestock Nutrition

Alfalfa for the compositional analysis was obtained from 5 replicated field sites across the United States from second cut forage. Four commercially available varieties were also grown at each site (12 commercial varieties in total). Forage samples were collected from all plots and analyzed for nutritional components.

Compositional analyses of forage samples included protein, fat, ash, moisture, ADF, NDF, lignin, amino acids, Ca, Cu, Fe, Mg, Mn, P, N, K, Na, Zn and carbohydrates. Events J101, J163 and J101 x J163 were compared to a non-transgenic control and further compared to 12 commercial varieties. Statistical differences were observed with respect to select amino acids, NDF and sodium however the variation was attributed to normal variability rather than the inserted novel traits.

CFIA has determined that Roundup Ready alfalfa containing event J101, J163 or J101 x J163 is nutritionally equivalent to non-transgenic alfalfa and similar to commercial varieties grown in North America.

2. Potential Impact on Livestock and Workers/By-standers

EPSPS is an enzyme present in many foods 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 events J101 and J163 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 conditions in the gastrointestinal tract. Additionally, a mouse acute oral toxicity study showed a NOEL of 475 mg/kg CP4 EPSPS. Based on the information provided by Monsanto Canada, CP4 EPSPS is unlikely to be a novel toxin or allergen.

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.

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 the release in Canada or elsewhere of alfalfa events J101 and J164, their descendants, or products derived there from, Monsanto Canada, Inc. must immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of the proposed feed use and environmental release and will re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of this alfalfa line.

VII. Regulatory Decision

Based on the review of the data, information and stewardship plan submitted by Monsanto Canada, Inc., and through comparisons of Roundup Ready® alfalfa events J101 and J163 with unmodified alfalfa counterparts, the PBO of the Plant Products Directorate, CFIA, has concluded that the novel gene and its corresponding trait does not confer to these plants any characteristic that would result in unintended environmental effects following unconfined release.

Based on the review of submitted data and information, the Animal Feed Division of the Animal Health Directorate has concluded that the novel trait does not in itself raise any concerns regarding the safety or nutritional composition of events J101 and J163. Alfalfa forage and its by-products are currently listed in Schedule iv of the Feeds Regulations and are therefore approved for use in livestock feeds in Canada. Events J101 and J163 have been assessed and found to be substantially equivalent to traditional alfalfa varieties. Events J101 and J163 and its by-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 alfalfa events J101 and J163 is therefore authorized as of July 28, 2005. Any other alfalfa varieties and intra-specific hybrids resulting from the same transformation event and all their descendants, may also be released into the environment and used as livestock feed, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown alfalfa, in terms of their potential environmental impact and livestock feed safety and efficacy.

The alfalfa events J101 and J163 are 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 alfalfa events J101 and J163. 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 Biosecurity Directorate and the Animal Health Directorate. For further information, please contact the Plant Biosafety Office or the Animal Feed Division at:

Plant Biosafety Office
Plant Health and Biosecurity Directorate
59 Camelot Drive
Ottawa, Ontario K1A 0Y9
613-225-2342

Animal Feed Division
Animal Health Directorate
59 Camelot Drive
Ottawa, Ontario K1A 0Y9
613-225-2342

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