Decision Document DD2014-106
Determination of the Safety of Monsanto Canada Inc. and Forage Genetic International's Alfalfa (Medicago sativa L.) Event KK179

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October 06, 2014

This Decision Document has been prepared to explain the regulatory decisions reached under Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits, its companion document BIO2005-02 - The Biology of Medicago sativa L. (alfalfa) and Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Biosecurity Directorate, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate and the Animal Feed Division of the Animal Health Directorate, has evaluated information submitted by Monsanto Canada Inc. and Forage Genetics International. This information is in regard to the reduced lignin alfalfa event KK179. 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 alfalfa varieties currently grown and permitted to be used as livestock feed in Canada.

Taking into account these evaluations, unconfined release into the environment and use as livestock feed of alfalfa event KK179 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate, respectively, as of October 06, 2014. Any alfalfa lines derived from alfalfa event KK179 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, (iii) it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to alfalfa varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and (iv) the novel gene is expressed at a level similar to that of the authorized line.

Alfalfa event KK179 is subject to the same phytosanitary import requirements as unmodified alfalfa varieties. Alfalfa event KK179 is required to meet the requirements of other jurisdictions, including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

Please note that the livestock feed and environmental safety assessments of novel feeds and PNTs are critical steps in the potential commercialization of these plant types. Other requirements, such as the food assessment of novel foods by Health Canada, have been addressed separately from this review.

This bulletin was created by the Canadian Food Inspection Agency. For further information, please contact the Animal Feed Division by visiting the contact page.

Table of contents

I. Brief Identification of the Modified Plant

Designation of the Modified Plant: Alfalfa event KK179
OECD Unique Identifier MON-ØØ179-5

Applicant: Monsanto Canada Inc. and Forage Genetics International

Plant Species: Alfalfa (Medicago sativa L.)

Novel Traits: Reduced lignin

Trait Introduction Method: Agrobacterium-mediated transformation

Intended Use of the Modified Plant: Alfalfa event KK179 is intended to be grown for traditional alfalfa livestock feed uses. Alfalfa event KK179 is not intended to be grown outside the normal production area for alfalfa in Canada.

II. Background Information

Monsanto Canada Inc. and Forage Genetics International have developed an alfalfa (Medicago sativa L.) event with reduced lignin compared to conventional alfalfa at the same stage of growth. Alfalfa event KK179 was developed by Monsanto Canada Inc. and Forage Genetics International using recombinant DNA technology, resulting in the introduction of a suppression cassette made up of gene sequences from the alfalfa enzyme caffeoyl CoA 3-O-methyltransferase (CCOMT). Transcription of this CCOMT suppression cassette results in reduced expression of the endogenous alfalfa CCOMT through a process known as RNA interference (RNAi). CCOMT is a key enzyme in the lignin biosynthetic pathway and its suppression results in reduced levels of guaiacyl lignin subunits (G lignin) and, correspondingly, reduced levels of total lignin compared to conventional alfalfa at the same stage of growth. Alfalfa event KK179 was developed to allow growers greater flexibility in harvest timing in order to better manage forage quality.

Monsanto Canada Inc. and Forage Genetics International have provided information on the identity of alfalfa event KK179, a detailed description of the transformation method and information on the insert copy number and intactness, northern blot analyses as evidence of the endogenous CCOMT gene suppression in the plant, and the role of the inserted suppression cassette and regulatory sequences.

Alfalfa event KK179 was field tested at 9 locations in the United States (US) and 1 location in Southern Ontario between 2010 and 2012. The US locations of these trials share similar environmental and agronomic conditions to alfalfa production areas in Canada and were considered representative of major Canadian alfalfa growing regions. An unmodified control alfalfa variety which shares the same genetic background as alfalfa event KK179 but has not been modified was included in the trials to act as a comparator for alfalfa event KK179. Several reference alfalfa varieties were also included in these trials to establish ranges of comparative values that are typical of currently grown alfalfa varieties.

Agronomic characteristics of alfalfa event KK179, such as seedling emergence, early season vigour, lodging, crop growth stage, forage yield, fall plant height, spring vigour, spring stand recovery and spring stand count, were compared to those of the unmodified control alfalfa variety and to the ranges established from the reference alfalfa varieties.

Nutritional components of alfalfa event KK179 forage, such as proximates (protein, fat, moisture and ash), carbohydrates (calculated), acid detergent fiber, neutral detergent fiber, acid detergent lignin, amino acids, minerals, secondary metabolites and anti-nutrients, were compared to those of the unmodified control alfalfa variety and to the range established from the reference alfalfa varieties.

The Plant and Biotechnology Risk Assessment (PBRA) Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in Directive 94-08 (Dir94-08) - Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.

The PBRA Unit has considered:

  • the potential for alfalfa event KK179 to become a weed of agriculture or to be invasive of natural habitats;
  • the potential for gene flow from alfalfa event KK179 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive;
  • the potential for alfalfa event KK179 to become a plant pest;
  • the potential impact of alfalfa event KK179 and its gene products on non-target organisms, including humans; and
  • the potential impact of alfalfa event KK179 on biodiversity.

The Animal Feed Division (AFD) of the 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 Section 2.6 - Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards.

The AFD has considered both intended and unintended effects and similarities and differences between alfalfa event KK179 and unmodified alfalfa varieties relative to the safety and efficacy of feed ingredients derived from alfalfa event KK179 for their intended purpose, including:

  • the potential impact of alfalfa event KK179 on livestock nutrition; and
  • the potential impact of alfalfa event KK179 on animal health and human safety, as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.

The AFD has also considered whether feeds derived from alfalfa event KK179 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

III. Description of the Novel Traits

1. Development Method

Alfalfa event KK179 was developed through Agrobacterium-mediated transformation of alfalfa cells using a transformation plasmid that includes 2 separate transfer DNAs (T-DNAs). The first T-DNA, designated as T-DNA I, contains the CCOMT suppression cassette. The second T-DNA, designated T-DNA II, contains the nptII expression cassette, encoding the NPTII protein, which confers resistance to kanamycin. During transformation, both T-DNAs were inserted into the alfalfa genome at unlinked loci, with T-DNA II functioning as a marker gene for in vitro selection of transformed plantlets. Through subsequent traditional alfalfa breeding methods, the unlinked insertions of T-DNA I and T-DNA II segregated, and a subset of transformed plants that contained T-DNA I (the CCOMT suppression cassette) but did not contain T-DNA II were identified. Alfalfa event KK179 was identified as a successful transformant and was chosen for further development.

2. Reduced Levels of Lignin

Alfalfa event KK179 is designed to reduce the amount of lignin that accumulates in forage compared to conventional alfalfa harvested at the same stage. Alfalfa event KK179 was produced by insertion of the CCOMT gene suppression cassette, which contains sequences from the alfalfa CCOMT gene. Transcription of the CCOMT suppression cassette results in the reduced expression of the endogenous alfalfa CCOMT transcript, through the RNAi process. This is anticipated to result in lower CCOMT protein levels, which leads to reduced production of G lignin compared to conventional alfalfa at the same stage of growth. The reduction in G lignin subunit production leads to reduced accumulation of total lignin, as confirmed through measurement of acid detergent lignin (ADL) by commercial forage testing methods.

The CCOMT suppression cassette in alfalfa event KK179 contains a promoter that drives expression in the vascular tissue of roots, stems and petioles, which corresponds with sites of lignin deposition in maturing plants. Northern blot data demonstrated a reduction in the level of CCOMT RNA in alfalfa event KK179 compared to the unmodified control alfalfa variety in both forage and root tissue.

Expression of the CCOMT suppression cassette does not result in the expression of novel proteins. Furthermore, small noncoding RNAs (ncRNAs), such as those present in alfalfa event KK179, are present in all plants and animals where they play central roles in endogenous gene regulation and response to exogenous DNA. The ncRNAs in alfalfa event KK179 are expected to function in a similar manner. Finally, the systemic absorption of exogenous ncRNAs in crops by livestock or human bystanders is considered to be unlikely based on information in the scientific literature.

3. Stable Integration into the Plant Genome

Molecular characterization of alfalfa event KK179 by Southern blot analyses demonstrated that alfalfa event KK179 contains 1 intact copy of the CCOMT suppression cassette inserted at a single site in the alfalfa genome. No additional elements, including intact or partial DNA fragments of the suppression cassette, T-DNA II or backbone sequences from the plasmid vector, linked or unlinked to the intact insert, were detected in alfalfa event KK179. Sequencing of the insert and adjacent genomic DNA sequence in alfalfa event KK179 confirmed the integrity of the inserted CCOMT suppression cassette and identified the 5' and 3' insert-to-genomic DNA junctions.

The stability of the insert within alfalfa event KK179 was verified by Southern blot analysis of progeny from 4 generations. The inheritance pattern of the insert across multiple segregating generations of alfalfa event KK179 showed that the insert segregates according to Mendelian rules of inheritance for a single genetic locus.

IV. Criteria for the Environmental Assessment

1. Potential for Alfalfa Event KK179 to Become a Weed of Agriculture or be Invasive of Natural Habitats

The CFIA biology document Bio2005-02: The Biology of Medicago sativa L. (Alfalfa) shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Alfalfa plants can grow as volunteers in cultivated fields in the seasons following an alfalfa crop, but they are usually eliminated by soil cultivation or the use of herbicides. According to the information provided by Monsanto Canada Inc. and Forage Genetics International, alfalfa event KK179 was determined not to be significantly different from unmodified alfalfa varieties in this respect.

The CFIA evaluated data submitted by Monsanto Canada Inc. and Forage Genetics International on the reproductive biology and life history traits of alfalfa event KK179. As previously mentioned, this event was field tested at 9 locations in the US and 1 location in Southern Ontario between 2010 and 2012. It was determined that the US locations share similar environmental and agronomic conditions to the alfalfa growing regions of Canada. In particular, several sites were considered to be representative of the alfalfa growing regions in Quebec and Ontario, where alfalfa event KK179 is more likely to be grown as dairy cattle forage. During the field trials, alfalfa event KK179 was compared to the unmodified control alfalfa variety. Reference alfalfa varieties were also included in these trials to establish ranges of comparative values that are representative of currently grown alfalfa varieties. Phenotypic and agronomic traits were evaluated, including seedling emergence, early season vigour, lodging, crop growth stage, forage yield, fall plant height, spring vigour, spring stand recovery and spring stand count. The results showed no biologically meaningful differences between alfalfa event KK179 and the unmodified control alfalfa variety and support a conclusion of phenotypic and agronomic equivalence to currently grown alfalfa varieties.

Seed dormancy and germination of scarified and non-scarified seeds from alfalfa event KK179 were evaluated under a range of temperature regimes. Although instances of statistically significant differences were observed between alfalfa event KK179 and the unmodified control alfalfa variety, values for alfalfa event KK179 were within the ranges established from the reference alfalfa varieties or within the ranges recorded in published literature. Therefore, the statistical analysis of these observations showed no biologically meaningful differences between alfalfa event KK179 and the unmodified alfalfa control variety. The results indicate that the introduction of the novel trait did not impact the germination or dormancy of the alfalfa seed.

Alfalfa event KK179 was exposed to abiotic stressors, including drought, flood, frost, hail, heat, nutrient deficiency, soil compaction and wind damage, during the agronomic characteristics studies. No trend in increased or decreased susceptibility to these abiotic stressors was observed in alfalfa event KK179 compared to the unmodified control alfalfa variety.

The susceptibility of alfalfa event KK179 to various alfalfa pests and pathogens was evaluated in the field at the same locations as the agronomic characteristics studies as well as in greenhouse experiments (further detail provided below in Section IV, part 3: Potential for Alfalfa Event KK179 to Become a Plant Pest). No trend in increased or decreased susceptibility to pests and pathogens was observed in alfalfa event KK179 compared to the unmodified control alfalfa variety.

No competitive advantage was conferred to alfalfa event KK179, as the reproductive characteristics, growth characteristics and tolerance to abiotic and biotic stresses of alfalfa event KK179 were comparable to those of the unmodified control alfalfa variety.

The novel trait has no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that alfalfa event KK179 has no altered weediness or invasiveness potential in Canada compared to currently grown alfalfa varieties.

2. Potential for Gene Flow from Alfalfa Event KK179 to Sexually Compatible Plants for Which Hybrid Offspring May Become More Weedy or More Invasive

According to the CFIA biology document Bio2005-02: The Biology of Medicago sativa L. (Alfalfa), there is a negligible risk for interspecific hybridization of alfalfa with any other species in North America. The one known relative of alfalfa in Canada is the naturalized species known as black medic (M. lupulina), but it has been reported that hybrids between alfalfa and black medic are considered impossible or exceedingly unlikely to occur in nature. This information, together with the fact that the novel trait has no intended effects on alfalfa reproductive biology, leads the CFIA to conclude that there is minimal potential for gene flow from alfalfa event KK179 to related species in Canada.

Monsanto Canada Inc. and Forage Genetics International also evaluated the viability, diameter and morphology of pollen from alfalfa event KK179. No statistically significant differences were detected between alfalfa event KK179 and the unmodified control alfalfa variety for percent viable pollen or pollen grain diameter. Furthermore, no visual differences in general pollen morphology were observed between alfalfa event KK179 and the unmodified control alfalfa variety. Therefore, the introduction of the novel trait did not impact the viability and morphology of pollen in alfalfa event KK179.

The reduced lignin trait in alfalfa event KK179 has no intended or observed effects on weediness or invasiveness that would confer a competitive advantage to the hybrid offspring. Furthermore, alfalfa managed for forage production is typically harvested before 10% bloom both to optimize the number of cuts that can be achieved in a growing season, and due to decreased forage quality after flowering from increasing total lignin content and reduction in crude protein and a corresponding rise in ADF. The same management and harvest practices are therefore anticipated for alfalfa event KK179. Thus, no substantial increase in gene flow between alfalfa event KK179 and other alfalfa fields or feral alfalfa populations is expected, compared to unmodified alfalfa varieties.

This information led the CFIA to conclude that gene flow from alfalfa event KK179 to other alfalfa plants is possible but would not result in increased weediness or invasiveness of the resulting progeny.

3. Potential for Alfalfa Event KK179 to Become a Plant Pest

According to the CFIA biology document Bio2005-02: The Biology of Medicago sativa L. (Alfalfa), alfalfa is not considered a plant pest in Canada.

The potential for the reduced lignin trait introduced into alfalfa event KK179 to have an impact on plant pest potential (i.e. the potential for the plant to harbour new or increased populations of pathogens or pests) was considered. Lignin plays a role in plant defense against pathogens and pests and the CCOMT enzyme has been implicated in disease resistance. As such, the susceptibility of alfalfa event KK179 to various alfalfa pests and pathogens was evaluated in the field at the same locations as the agronomic characteristics studies. The stressors observed included alfalfa caterpillar, alfalfa leafminer, alfalfa weevil, aphid, armyworm, bean leaf beetle, blister beetle, cutworms, grasshopper, green cloverworm, Japanese beetle, lygus bug, plant bug, meadow spittlebug, pea aphid, potato leafhopper, southern corn rootworm beetle, spider mite, thrips, anthracnose, bacterial wilt, black stem, damping-off, downy mildew, Fusarium wilt, leaf spots, root rot (including Phytophthora), Sclerotinia crown and stem rot, stem nematode and Verticillium wilt. No qualitative differences were observed between alfalfa event KK179 and the unmodified control alfalfa variety for any of the 225 observations for plant damage caused by arthropods and for any of the 222 observations for plant damage caused by diseases.

Monsanto Canada Inc. and Forage Genetics International quantitatively assessed alfalfa weevil and potato leafhopper damage in the field at three of the same locations as the agronomic characteristics studies. Although instances of significant differences were detected between alfalfa event KK179 and the unmodified control alfalfa variety for alfalfa weevil and potato leafhopper damage in the individual-site analyses, there was no consistent trend observed across observation times or sites. Therefore, the differences were not considered biologically meaningful in terms of plant pest potential.

Greenhouse experiments were also conducted to compare the responses of alfalfa event KK179 and the unmodified control alfalfa variety to bacteria wilt, Fusarium wilt, Verticillium wilt, anthracnose, Aphanomyces (races 1 and 2), Phytophthora, alfalfa stem nematode and northern root-knot nematode. No statistically significant differences were detected between alfalfa event KK179 and the unmodified control alfalfa variety in percent resistance for any of the pathogens or nematodes tested.

The abundance of pest arthropods in alfalfa event KK179 plots was evaluated in the field at three of the same locations as the agronomic characteristics studies. The pests observed included alfalfa looper, alfalfa weevil, aphid, armyworm, blister beetle, false chinch bug, garden webworm, green cloverworm, lygus bug, meadow spittlebug, potato leafhopper, soybean looper, and thrips. Although instances of statistically significant differences were observed between alfalfa event KK179 and the unmodified control alfalfa variety for some pests in the individual-site analyses, there was no consistent trend observed across observation times or sites. Therefore, the differences were not considered biologically meaningful.

The CFIA has therefore concluded that alfalfa event KK179 does not display any altered plant pest potential compared to the alfalfa varieties currently grown in Canada.

4. Potential Impact of Alfalfa Event KK179 on Non-Target Organisms, Including Humans

The reduced lignin trait introduced in alfalfa event KK179 is unrelated to a potential impact on non-target organisms.

The CCOMT suppression cassette introduced into alfalfa event KK179 does not result in the expression of a novel protein. Therefore, there is no risk of new protein toxins or allergens being introduced into alfalfa event KK179. The safety assessment of alfalfa event KK179 focused on ncRNAs and the suppression of the expression of endogenous CCOMT enzyme levels. The production of ncRNA for naturally occurring endogenous RNAi is common in plants and animals, and the CCOMT suppression cassette in alfalfa event KK179 was specifically designed to target the alfalfa plant. Therefore, the alfalfa CCOMT suppression cassette in alfalfa event KK179 is not expected to mediate the inhibition of gene expression in organisms interacting with alfalfa event KK179. Based on the evidence provided by Monsanto Canada Inc. and Forage Genetics International, no negative impacts resulting from exposure of organisms to ncRNA expressed from the CCOMT suppression cassette in alfalfa event KK179 are expected.

Compositional analyses showed that with the exception of the intended decrease in G lignin subunit and total lignin, the nutritional composition of alfalfa event KK179 is equivalent to that of the unmodified control alfalfa variety (see Section V, part 1: Potential Impact of Alfalfa Event KK179 on Livestock Nutrition). Furthermore, the total lignin content in alfalfa event KK179 is within the range established from the reference alfalfa varieties grown at the same time. Therefore, it is very unlikely that the introduction of the novel trait may have caused unintended changes to the composition of alfalfa event KK179 tissues that would negatively impact organisms interacting with the plants.

Field evaluations of alfalfa event KK179 did not show any increased resistance to insect pests or pathogens compared to the unmodified control alfalfa variety or any biologically meaningful differences in the abundance of pest arthropods (see Section IV, part 3: Potential for Alfalfa Event KK179 to Become a Plant Pest).

The abundance of beneficial arthropods in alfalfa event KK179 plots was evaluated in the field in three of the same locations as the agronomic characteristics studies. The arthropods observed included spiders, chalcid wasps, damsel bugs, ladybird beetles, lacewings, nabids, and parasitic wasps. Although instances of statistically significant differences were observed between alfalfa event KK179 and the unmodified control alfalfa variety for some of these beneficial arthropods in the individual-site analyses, these differences were not considered to be biologically significant because there was no consistent trend observed across observation times or sites and the arthropod numbers observed on alfalfa event KK179 were within the ranges of values established from the reference alfalfa varieties.

Collectively, these information elements indicate that the interactions between alfalfa event KK179 and the populations of animals and microorganisms interacting with alfalfa crops will be similar compared to currently grown alfalfa varieties.

The CFIA has therefore determined that the unconfined release of alfalfa event KK179 in Canada will not result in altered impacts on non-target organisms, including humans, compared to currently grown alfalfa varieties.

5. Potential Impact of Alfalfa Event KK179 on Biodiversity

Alfalfa event KK179 expresses no novel phenotypic characteristics that would extend its geographic range beyond the current range of alfalfa production in Canada. Since alfalfa does not outcross to wild relatives in Canada there will be no transfer of the novel trait to other species in unmanaged environments. Alfalfa event KK179 is unlikely to cause adverse effects on non-target organisms and does not display increased weediness, invasiveness or plant pest potential. It is therefore unlikely that alfalfa event KK179 will have any direct or indirect effects on biodiversity, in comparison to the effects that would be expected from the cultivation of the alfalfa varieties that are currently grown in Canada.

The CFIA has concluded that the introduced gene suppression cassette and its corresponding novel trait do not confer to alfalfa event KK179 any characteristic that would result in unintended environmental effects following unconfined release.

The CFIA has therefore concluded that the impact on biodiversity of alfalfa event KK179 is unlikely to be different from that of alfalfa varieties that are currently grown in Canada.

V. Criteria for the Livestock Feed Assessment

The AFD considered nutrient and anti-nutrient profiles; the safety of feed ingredients derived from alfalfa event KK179, including the presence of gene products, residues and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed; and whether feeds derived from alfalfa event KK179 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

1. Potential Impact of Alfalfa Event KK179 on Livestock Nutrition

Nutrient and anti-nutrient composition

The nutritional equivalence of alfalfa event KK179 to the unmodified control alfalfa variety and 14 reference alfalfa varieties was determined from 6 replicated US field trials during the 2011 growing season. The field plots were established from plants started in a greenhouse. At each site, plants were transplanted in a randomized complete block design with 4 replications (plots) each of alfalfa event KK179, the unmodified control alfalfa variety and 4 reference alfalfa varieties. Forage samples were collected from each plot at the first cutting between 1 and 10% bloom stages of the plant growth. The levels of lignin subunits and total lignin in the samples were initially analysed to examine the intended reduced lignin trait of alfalfa event KK179. Further analyses of ash, fat, moisture, protein, carbohydrates (calculated), acid detergent fiber (ADF), neutral detergent fiber (NDF), acid detergent lignin (ADL), minerals (Ca, Cu, Fe, Mg, Mn, P K, Na and Zn) and amino acids were conducted. Anti-nutrients analysed were daidzein, glycitein, genistein, coumesterol, formononetin, biochanin A and saponins (total bayogenin, total hederagenin, total medicagenic acid, total soyasapogenol B, total soyasapogenol E, total zanhic acid and total saponins). The secondary metabolites; p-coumaric acid, ferulic acid, sinapic acid; total polyphenols, free phenylalanine and canavanine were also analyzed to evaluate the effect of CCOMT suppression on the lignin biosynthetic pathway and cell wall-associated metabolites, as recommended by the OECD consensus document for new varieties of alfalfa – PDF (257 kb).

Composition data were analyzed statistically using analysis of variance and statistical differences between alfalfa event KK179 and the unmodified control alfalfa variety were identified (P<0.05). The biological relevance of any significant difference between alfalfa event KK179 and the unmodified control alfalfa variety was assessed within the natural variability of the values obtained from the reference alfalfa varieties and also from the published scientific literature. The mean value of G lignin in alfalfa event KK179 was statistically significantly reduced (68.1 μmol/g cell wall residue (CWR)), compared to the unmodified control alfalfa variety (83.7 μmol/g CWR). As such, the mean ADL in alfalfa event KK179 (5.39% dwt) was statistically significantly lower than the unmodified control alfalfa variety (6.93% dwt), when cut at the same stage of growth. All means were, however, within the range of the values observed in the reference alfalfa varieties and the published scientific literature.

Except for ash, there were no statistically significant differences between alfalfa event KK179 and unmodified control alfalfa variety cut at the same stage of growth for crude fat, ash, protein, carbohydrates (calculated), ADF and NDF. The mean ash values were within the range of the values observed in the reference alfalfa varieties and the published scientific literature; and therefore the differences were not considered biologically relevant. There was a numerical decrease in ADL in alfalfa event KK179 compared to unmodified control alfalfa variety. There were no statistically significant differences between the concentrations of amino acids and minerals analysed in alfalfa event KK179 when compared to the unmodified control alfalfa variety. No statistically significant differences were also observed between alfalfa event KK179 and the unmodified control alfalfa variety for the secondary metabolites, p-coumaric acid, sinapic acid, total polyphenols and free phenylalanine. Statistically significant differences were observed between alfalfa event KK179 and the unmodified control alfalfa variety for canavanine and ferulic acid, however the mean values were within the range of the reference alfalfa varieties and literature values, and therefore the differences were not considered biologically relevant. There were no statistically significant differences between the mean concentrations of total bayogenin, hederagenin, medicagenic acid, soyasapogenol B, soyasapogenol E, zanhic acid and total saponins from alfalfa event KK179 compared to the unmodified control alfalfa variety. The secondary metabolite, sinapic acid and antinutrients, daidzein, glycitein, genistein, coumesterol, formononetin, biochanin A had more than 50% of the observations below the assay limit of quantification in both alfalfa event KK179 and the unmodified control alfalfa variety and therefore were excluded from statistical analyses.

Animal performance

The nutritional efficacy of alfalfa event KK179 hay compared to the unmodified control alfalfa variety was evaluated in 22 lambs (11 males and 11 females) over a 28-d feeding trial. Both hay sources were obtained from a second cutting (at 1-10% bloom stage of plant growth) of a 2011 alfalfa production in its first year of establishment. The diet was 100% alfalfa hay with a free-choice mineral supplement. Alfalfa hay intake was not different in lambs fed alfalfa event KK179 when compared to those fed the unmodified control alfalfa hay. Initial and final body weights were not different in lambs fed alfalfa event KK179 compared to unmodified control alfalfa hay. Male lambs fed alfalfa event KK179 hay showed increased body gain, average daily gain and gain:feed ratio compared to male lambs fed the unmodified control alfalfa hay. Average body temperature and blood serum chemistry components during the treatment period were similar in lambs fed alfalfa event KK179 hay compared to those fed the unmodified control alfalfa hay. No abnormalities of tissues or mortality were related to the hay treatments during the feeding period. In summary, the growth of lambs consuming alfalfa event KK179 hay was comparable to lambs consuming unmodified control alfalfa hay. Lambs consuming alfalfa event KK179 had comparable general health to those consuming the unmodified alfalfa hay.

Conclusion

It was concluded based on the evidence provided by Monsanto Canada Inc. and Forage Genetics International that with the exception of the intended decrease in G lignin subunit and total lignin, the nutritional composition of alfalfa event KK179 is similar to that of the unmodified control alfalfa variety, cut at the same growth stage. Given that the total lignin content in alfalfa event KK179 is within the natural range of reference alfalfa varieties currently grown and will be used in the same way as conventional alfalfa, feed ingredients therefore derived from alfalfa event KK179 are considered to meet the present ingredient definition for alfalfa in Schedule IV of the Feeds Regulations.

2. Potential Impact of Alfalfa Event KK179 on Animal Health and Human Safety as it Relates to the Potential Transfer of Residues into Foods of Animal Origin, and Worker/Bystander Exposure to the Feed.

Alfalfa event KK179 has reduced lignin compared to conventional alfalfa at the same growth stage as a result of the insertion of a CCOMT suppression cassette whose expression results in RNAi-mediated suppression of the endogenous alfalfa CCOMT gene. A weight-of-evidence approach was used to assess the impacts of alfalfa event KK179 on the following potential hazards relative to the safety of feed ingredients derived from these events:

CCOMT RNA products

Expression of the CCOMT suppression cassette does not result in production of novel proteins. Therefore, there is no risk of new protein toxins or allergens being introduced into alfalfa events KK179. The livestock feed safety assessment of alfalfa event KK179 focused on the safety of suppression of the expression of endogenous CCOMT enzyme levels and the small noncoding RNAs. Suppression of the expression of endogenous CCOMT enzyme levels in alfalfa event KK179 was not considered to present a risk to human or animal health or the environment since there was no evidence of association with a toxic mode of action and there was no evidence that reduction of CCOMT enzyme levels would impact endogenous alfalfa toxin or allergen levels. Small noncoding RNAs present in alfalfa event KK179 were not considered to present a risk to human or animal health or the environment because bioinformatics analyses demonstrated that the DNA sequences inserted into alfalfa event KK179 did not have significant similarity to protein-coding genomic regions of livestock animals or humans thus were not expected to affect livestock or human bystander protein expression. Also, a lack of significant similarity was observed between DNA sequences inserted into alfalfa event KK179 and protein-coding genomic regions in alfalfa, other than the CCOMT gene, suggesting the DNA sequences were not expected to affect expression of other alfalfa genes. Finally, the systemic absorption of exogenous small noncoding RNAs in crops by livestock or human bystanders is considered to be unlikely based on existing biological barriers.

Conclusions

It was concluded, based on the evidence provided by Monsanto Canada Inc. and Forage Genetics International, that the novel reduced lignin trait will not confer to alfalfa event KK179 any characteristic that would raise concerns regarding the feed safety of alfalfa event KK179. Feed ingredients derived from alfalfa event KK179 are considered to meet present feed ingredient definitions for alfalfa.

VI. New Information Requirements

If at any time, Monsanto Canada Inc. and Forage Genetics International become aware of any new information regarding risk to the environment, livestock or human health, which could result from release or livestock feed use of alfalfa event KK179 or lines derived from it, Monsanto Canada Inc. and Forage Genetics International are required to immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of alfalfa event KK179 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 alfalfa event KK179.

VII. Regulatory Decision

Based on the review of the data and information submitted by Monsanto Canada Inc. and Forage Genetics International and input from other relevant scientific sources, the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has concluded that the unconfined environmental release of alfalfa event KK179 does not present altered environmental risk when compared to alfalfa varieties that are currently grown in Canada.

Based on the review of the data and information submitted by Monsanto Canada Inc. and Forage Genetics International and input from other relevant scientific sources, the Animal Feed Division of the Animal Health Directorate, CFIA, has concluded that the reduced G lignin production trait will not confer to alfalfa event KK179 any characteristic that would raise concerns regarding the safety or nutritional composition of alfalfa event KK179. Its byproducts are currently listed in schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Alfalfa event KK179 has been found to be as safe and nutritious as currently and historically grown alfalfa varieties. Alfalfa event KK179 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 and use as livestock feed of alfalfa event KK179 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of October 6, 2014. Any alfalfa lines derived from alfalfa event KK179 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, (iii) it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to alfalfa varieties that are currently grown and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety, respectively and (iv) the novel gene is expressed at a level similar to that of the authorized line.

Alfalfa event KK179 is subject to the same phytosanitary import requirements as unmodified alfalfa varieties. Alfalfa event KK179 is required to meet the requirements of other jurisdictions, including but not limited to, the Food & Drugs Act and the Pest Control Products Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of alfalfa event KK179.

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