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Decision Document DD95-03:
Determination of Environmental Safety of
Pioneer Hi-Bred International Inc.'s
Imidazolinone-Tolerant Canola

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and its companion document Dir94-09 The Biology of Brassica napus L. (Canola/Rapeseed), and the proposed guidelines Pro94-04 Guidelines for the Assessment of Plants with Novel Traits as Livestock Feed.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division, has evaluated information submitted by Pioneer Hi-Bred International Inc. regarding three imidazolinone-tolerant canola lines, NS738, NS1471, and NS1473. It has been determined that these plants with novel traits do not present altered environmental interactions when compared to currently commercialized canola varieties in Canada.

The Feed Section of the Animal Health and Production Division (CFIA), has also evaluated information submitted by Pioneer Hi-Bred International Inc. regarding the imidazolinone-tolerant canola line NS1471 and have determined that it is substantially equivalent to canola currently approved as livestock feed.

Unconfined release into the environment of the lines NS738, NS1471, and NS1473, and other B. napus lines derived from them, but without the introduction of any other novel trait, is therefore considered safe.

Feed use of the NS1471 line, and other B. napus lines derived from it, but without the introduction of any other novel trait, is also considered safe.

Table of Contents

1. Brief Identification of the Plants with Novel Traits (PNTs)
2. Background Information
3. Description of the Novel Traits
   1. Imidazolinone Tolerance
   2. Development Method
   3. Stable Expression
4. Assessment Criteria for Environmental Safety
   1. Potential of the PNTs to become Weeds of Agriculture or to be Invasive of Natural Habitats
   2. Potential for Gene Flow to Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
   3. Altered Plant Pest Potential
   4. Potential Impact on Non-Target Organisms
   5. Potential Impact on Biodiversity
5. Nutritional Assessment Criteria as Livestock Feed
   1. Anti-Nutritional Factors
   2. Nutritional Composition of the PNTs
6. Regulatory Decision

I. Brief Identification of the Plants with Novel Traits (PNTs)

II. Background Information

Pioneer Hi-Bred International Inc. has developed three Brassica napus canola lines tolerant to imidazolinone herbicides, specifically to Cyanamid's Pursuit® herbicide, currently registered in Canada as a post-emergence herbicide for the control of various broadleaved and grass weeds. These B. napus lines, referred to as NS738, NS1471 and NS1473 in the present document, exhibit no significant injury when treated with Pursuit® at normal field application rates, and will allow the use of imidazolinone herbicides as post-emergence herbicides on canola crops, thus providing an alternative for weed control in canola production.

The development of each of NS738, NS1471 and NS1473 was based on chemically induced in vitro microspore modification. The herbicide tolerant phenotype results from the modification of genes belonging to the acetolactate synthase (ALS) multigene family, such that this enzyme is no longer affected by imidazolinones.

NS738 has been field tested in Canada since 1989 in the provinces of Alberta, Manitoba, Saskatchewan and Ontario. NS1471 and NS1473 have been tested since 1991. NS738 has gone through three years of public cooperative registration trials. NS1471 and NS1473 have gone through two years of cooperative trials. NS1471 was recommended for a two year interim registration by the Western Canada Canola/Rapeseed Recommending Committee (WCC/RRC) on April 6, 1995.

Pioneer Hi-Bred International Inc. has provided data on the identity of NS738, NS1471 and NS1473, a detailed description of the modification method and breeding history, information on the modified genes, the resulting protein and its mode of action and the stability of trait expression. References to scientific publications were also included.

Agronomic characteristics such as seed production, vegetative vigor, days to flowering, time to maturity, plant lodging, plant height, production of oil and protein, and disease susceptibilities were compared to those of unmodified B. napus counterparts.

Data to support the efficacy of NS1471 as a livestock feed was provided. A proximate analysis to include crude protein, crude fat, crude fiber, ash and gross energy were supplied for the whole seed, processed meal and oil content.

The Canadian Food Inspection Agency (CFIA) has reviewed the above information, in light of the assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08:

• potential of the PNTs to become weeds of agriculture or to 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 Feed Section of Animal Health and Production Division (CFIA), has also reviewed the above information in light of the assessment criteria for determining safety and efficacy of livestock feed, as described in Pro94-04:

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

III. Description of the Novel Traits

1. Imidazolinone Tolerance

• Imidazolinone herbicides are active against the enzyme acetolactate synthase (ALS), also known as acetohydroxyacid synthase (AHAS) or acetolactate pyruvate-lyase.
• This enzyme catalyzes the first step in the biosynthesis of the essential branched chain amino acids isoleucine, leucine and valine. Herbicide induced ALS inhibition results in a lethal decrease in protein synthesis. Unmodified canola is not tolerant to imidazolinones, while soybeans, peas, white beans and kidney beans are naturally tolerant to them.
• The genome of Brassica napus contains an ALS multigene family comprising five genes, two of which are constitutively expressed, share extensive homology with ALS genes from Arabidopsis thaliana and Nicotiana tabacum, and are assumed to encode the primary ALS activities necessary for plant growth and development.
• Modifications of ALS genes in various plant species can result in herbicide tolerant phenotypes and typically consist of one amino acid substitution, sufficient to alter the binding site for imidazolinones such that the herbicide no longer inactivates the ALS enzyme.
• Pioneer Hi-Bred International Inc. selected two B. napus lines, P1 and P2, with altered ALS enzymes not inhibited by the herbicide. The modifications in P1 and P2 occur at different and unlinked loci, presumably representing different ALS genes. Lines NS1471 and NS1473 contain both mutations, while line NS738 contains the P2 mutation only. The novel ALS enzyme in line P2 has been isolated and sequenced: it differs from the native gene by one amino acid substitution.
• The novel imidazolinone resistance is constitutively expressed and synthesis of the branched chain amino acids isoleucine, leucine and valine, is not affected by the ALS modifications in NS1471, NS1473 and NS738.

2. Development Method

• NS738, NS1471 and NS1473 were developed from the in vitro culture of microspores, from the B. napus cultivar Topas, mutagenized with ethyl nitrosourea and selected on imidazolinone containing culture medium. The regenerated haploid plantlets were recovered and treated with colchicine to induce chromosome duplication resulting in doubled-haploid plants.
• The two imidazolinone tolerant lines P1 and P2 were selected and crossed reciprocally then crossed with B. napus cultivars Topas and Regent, and the progeny subjected to repeated cycles of selfing and further breeding.

3. Stable Expression

• NS738, NS1471 and NS1473 are now several generations removed from the original modified materials, and still consistently show imidazolinone resistance.

IV. Assessment Criteria for Environmental Safety

CFIA evaluated data submitted by Pioneer Hi-Bred International Inc. from replicated testing at 21 different locations, representative of four different growing environments, on agronomic performance of NS738, NS1471 and NS1473. Based on this data, CFIA determined that seed production, vegetative vigor, days to flowering, time to maturity, plant lodging, plant height, production of oil and protein, and disease susceptibilities were within the normal range of unmodified B. napus counterparts. Valine, isoleucine and leucine contents of NS1471 were similar to those of current commercial canola cultivars, showing that ALS activity of the PNT was not affected by the mutation. Analyses of amino acid composition and of antinutritional factors (glucosinolates and erucic acid) also showed substantial equivalence to unmodified counterparts. The lines have no specific added genes that could confer them any ecological advantage over traditional canola plants.

These results collectively demonstrate that the mutations of the ALS enzyme in NS738, NS1471 and NS1473 did not likely result in any secondary effects impacting on environmental safety of these lines and no further safety considerations are anticipated.

1. Potential of the PNTs to become Weeds of Agriculture or to be Invasive of Natural Habitats

The biology of B. napus, described in Dir94-09, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information provided by Pioneer Hi-Bred, NS738, NS1471 and NS1473 were determined not to be different from their counterparts in this respect. No competitive advantage was conferred to imidazolinone-tolerant plants, other than that conferred by tolerance to imidazolinone.

Imidazolinone tolerance will not cause these plants to become more weedy or invasive. All commercial canola varieties are naturally tolerant to the ethametsulfuron methyl herbicide MUSTER® (DuPont), that also targets the ALS gene family, and these do not show invasive characteristics.

Imidazolinone-tolerant canola volunteers will not be controlled in subsequent crops when imidazolinones are used as the sole weed control tool.

Control of imidazolinone tolerant weeds, or imidazolinone tolerant canola as a volunteer weed in other crops or in fallow ground, can readily be achieved by the use of classes of herbicides other than imidazolinones, or by mechanical means.

The above considerations led CFIA to conclude that NS738, NS1471 and NS1473 have no altered weed or invasiveness potential compared to currently commercialized canola varieties.

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

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

Brassica napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of the species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum (Dir94-09). Studies show that introgression of the herbicide tolerance genes is most likely to occur with B. rapa, the other major canola species and an occasional weed of cultivated land especially in the eastern provinces of Canada.

If imidazolinone-tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless challenged by imidazolinones. This would only occur in managed ecosystems where imidazolinones are used for weed control. As with imidazolinone-tolerant B. napus, these herbicide tolerant individuals, should they arise, would be easily controlled using mechanical and other available chemical means. Hybrids, if they developed, could potentially result in the loss of imidazolinones as a tool to control these species. This, however, can be avoided by the use of sound crop management practices.

The above considerations led CFIA to conclude that gene flow from NS738, NS1471 and NS1473 to canola relatives is possible, but would not result in increased weediness or invasiveness of these relatives.

3. Altered Plant Pest Potential

The intended effect of the novel trait is unrelated to plant pest potential, and B. napus is not a plant pest in Canada (Dir94-09). In addition, agronomic characteristics and qualitative and quantitative composition of NS738, NS1471 and NS1473 were shown to be within the range of values displayed by currently commercialized B. napus varieties, leading to the conclusion that plant pest potential was not inadvertently altered.

CFIA has therefore determined that NS738, NS1471 and NS1473 did not display any altered pest potential.

4. Potential Impact on Non-Target Organisms

The novel trait results from single amino acid modifications of a B. napus enzyme, thus altering the herbicide binding sites without modification of metabolic abilities. Potential toxicity of this enzyme is therefore not a concern. Agronomic characteristics and nutritional considerations for livestock feed showed that the lines NS738, NS1471 and NS1473 are substantially equivalent to unmodified counterparts.

CFIA has therefore determined that the unconfined release of NS738, NS1471 and NS1473 will not result in altered impacts on interacting organisms, including humans, compared with currently commercialized counterparts.

5. Potential Impact on Biodiversity

NS738, NS1471 and NS1473 have no novel phenotypic characteristics which would extend their use beyond the current geographic range of canola production in Canada. Since outcross species are only found in disturbed habitats, transfer of the novel trait would not impact unmanaged environments.

CFIA has therefore concluded that the potential impact on biodiversity of NS738, NS1471 and NS1473 is equivalent to that of currently commercialized canola lines.

V. Nutritional Assessment Criteria as Livestock Feed

1. Anti-Nutritional Factors

95% Confidence Intervals were determined for the glucosinolate and erucic acid content of the meal and oil produced from NS1471 grown under a variety of conditions. These confidence intervals demonstrated that the PNT contained levels of these anti-nutritional factors below the prescribed standards for both the meal and oil fractions, i.e., < 30 micromoles glucosinolates per gram of dry meal and < 2% erucic acid in the oil.

2. Nutritional Composition of the PNTs

No statistical differences in nutritional composition, i.e., crude protein, crude fat, crude fibre, ash and gross energy content, were noted between the whole seed of NS1471 and those of current commercial canola cultivars. Valine, isoleucine and leucine contents of NS1471 were compared to that of current commercial canola cultivars to determine whether acetolactate synthase (ALS) activity of the PNT was affected by the mutation. No statistical difference in the content of these amino acids was noted. These results collectively demonstrate that the mutation of the ALS enzyme in NS1471 did not likely result in any secondary effects impacting on the composition or nutritional quality of the cultivar. Accordingly, NS1471 was judged to be substantially equivalent to traditional canola varieties in terms of nutritional composition.

VI. Regulatory Decision

Based on the review of data and information submitted by Pioneer Hi-Bred International Inc., including comparisons of NS738, NS1471 and NS1473 with unmodified B. napus counterparts, CFIA has concluded that the modified gene product and associated novel trait do not confer any intended or unintended ecological advantage to these lines. Should the trait be transferred through outcrossing to related plants, these would not result in any ecological advantage.

Based on the review of submitted data and information, the Feed Section of the Plant Health and Production Division has concluded that the novel trait does not in itself raise any concerns regarding the safety or nutritional composition of this line. Canola oil, seed and meal are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As NS1471 has been assessed and found to be substantially equivalent to traditional canola varieties, NS1471 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 of the lines NS738, NS1471, and NS1473, and other B. napus lines derived from them, but without the introduction of any other novel trait, is therefore considered safe.

Feed use of the NS1471 line, and other B. napus lines derived from it, but without the introduction of any other novel trait, is also considered safe.

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