DD2006-62: Determination of the Safety of BASF's Imidazolinone Tolerant CLEARFIELD™ Rice Events IMINTA 1 and IMINTA 4

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Issued: 2006-10

This Decision Document has been prepared to explain the regulatory decision reached under Directive 95-03 (Dir 95-03) "Guidelines for the Assessment of Novel Feeds: Plant Sources", including an environmental safety assessment carried out according to Directive 94-08 (Dir 94-08) "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits".

The Canadian Food Inspection Agency (CFIA), specifically the Feed Section of the CFIA, with advice from the Plant Biosafety Office of the CFIA, has evaluated information submitted by BASF regarding the imidazolinone tolerant rice events IMINTA 1 and IMINTA 4. The CFIA has determined that feed derived from these modified plants does not present a significant risk to the environment, nor do they present livestock feed safety concerns when compared to currently commercialized rice varieties in Canada.

Livestock feed use of rice events IMINTA 1 and IMINTA 4 is therefore authorized as of October 30, 2006. Any rice lines derived from IMINTA 1 and IMINTA 4 are also authorized for use in livestock feed, provided that: (i) no inter-specific crosses are performed, (ii) the intended use(s) are similar, (iii) it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown rice, in terms of their potential environmental impact and livestock feed safety.

Rice events IMINTA 1 and IMINTA 4 are subject to the same phytosanitary import requirements as their unmodified counterparts.

Please note that the assessment of livestock feed safety and environmental safety 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 Modified Plant

II. Background Information

III. Description and Assessment of the Novel Trait

  1. Development Method
  2. Imidazolinone Tolerance
  3. Stable Expression

IV. Criteria for the Environmental Assessment

  1. Potential of events IMINTA 1 and IMINTA 4 to Become Weeds of Agriculture or Invasive of Natural Habitats
  2. Potential for Gene Flow from events IMINTA 1 and IMINTA 4 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
  3. Altered Plant Pest Potential of events IMINTA 1 and IMINTA 4
  4. Potential Impact on Non-Target Organisms of events IMINTA 1 and IMINTA 4
  5. Potential Impact on Biodiversity of events IMINTA 1 and IMINTA 4

V. Nutritional Criteria Assessment as Livestock Feed

  1. Potential Impact of events IMINTA 1 and IMINTA 4 on Livestock Nutrition
  2. Potential Impact of events IMINTA 1 and IMINTA 4 on Livestock and Workers/By-standers

VI. New Information Requirements

VII. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation(s) of the Plant: CLEARFIELD™ rice events IMINTA 1 and IMINTA 4

Applicant: BASF Canada

Plant Species: Rice (Oryza sativa L.)

Novel Traits: Tolerance to imidazolinone herbicides

Trait Introduction Method: Chemically induced seed mutagenesis

Proposed Use of the Plant: Production of rice for livestock feed and human food

II. Background Information

BASF has developed rice events tolerant to imidazolinone herbicides. These rice events were developed to provide an alternative strategy for weed control.

The development of the rice events IMINTA 1 and IMINTA 4 was accomplished using chemically induced seed mutagenesis. The herbicide tolerance results from a single point mutation modification of the acetohydroxyacid synthase (AHAS) gene such that the resulting enzyme has a single amino acid substitution and is no longer affected by imidazolinone herbicides. Previously authorized rice events CL121, CL141, CFX52 and PWC16 also contain modified AHAS genes conferring tolerance to imidazolinone herbicides.

BASF has provided data on the identity of the rice events, a detailed description of the modification method and breeding history, information on the modified gene, the resulting protein and its mode of action and the stability of trait expression.

Events IMINTA 1 and IMINTA 4 were field tested in Argentina in 2002/2003 and 2003/2004.

Agronomic characteristics of events IMINTA 1 and IMINTA 4 such as yield, heading, days to flowering, seed weight, height, shattering and disease susceptibility were compared to those of unmodified rice counterparts.

Nutritional components of events IMINTA 1 and IMINTA 4 such as proximates, amino acids and fatty acids were compared with unmodified rice counterparts. Anti-nutritional factors were also determined.

The Feed Section, CFIA, with input from the Plant Biosafety Office, CFIA, has reviewed the above information. The following assessment criteria, as described in regulatory directives Dir 95-03 and Dir 94-08, were used to determine the safety and efficacy as livestock feed and the environmental safety of this novel feed:

  • potential impact of events IMINTA 1 and IMINTA 4 on livestock nutrition;
  • potential impact of events IMINTA 1 and IMINTA 4 on livestock and workers/by-standers;
  • potential of events IMINTA 1 and IMINTA 4 to become weeds of agriculture or invasive of natural habitats;
  • potential for gene flow from events IMINTA 1 and IMINTA 4 to wild relatives whose hybrid progeny may become more weedy or more invasive;
  • potential for events IMINTA 1 and IMINTA 4 to become plant pests;
  • potential impact of events IMINTA 1 and IMINTA 4 or their gene products on non-target species, including humans; and
  • potential impact of events IMINTA 1 and IMINTA 4 on biodiversity.

III. Description and Assessment of the Novel Trait

1. Development Method

Events IMINTA 1 and IMINTA 4 were derived by chemical-induced mutagenesis of seed of the rice variety IRGA 417 with sodium azide. Whole plant selection procedures for herbicide tolerance were used to select these events.

Events IMINTA 1 and IMINTA 4 are diploids (24 chromosomes), belonging to the genus and species Oryza sativa.

2. Imidazolinone Tolerance

Imidazolinone herbicides are active against the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS). AHAS is an enzyme found in bacteria, certain other micro-organisms and plants. This enzyme catalyses the first step in the biosynthesis of the essential branched chain amino acids isoleucine, leucine and valine. Herbicide induced AHAS inhibition results in a lethal decrease in protein synthesis. Unmodified rice is not tolerant to imidazolinone herbicides.

A single amino acid substitution in the AHAS enzyme was sufficient to alter the binding site for imidazolinones herbicides, resulting in the tolerant phenotype.

The novel imidazolinone tolerance is under the control of the native AHAS promoter and is believed to be constitutively expressed. Sequence information for the modified AHAS gene in IMINTA 1 and IMINTA 4 was submitted.

The tolerance to imidazolinone was demonstrated by comparison of the activity of the AHAS enzyme extracted from IMINTA 1 and IMINTA 4 rice plants to that of conventional type rice plants.

The levels of valine, leucine and isoleucine produced in rice are regulated by feedback inhibition of AHAS. BASF provided data to demonstrate that the modified AHAS shows similar feedback inhibition by valine and leucine as compared to unmodified AHAS. The modification of the AHAS does not affect feedback inhibition and hence, the regulation and levels of these amino acids.

Unlike known food allergens, AHAS is a minor protein in plant tissue, it is heat sensitive and trypsin susceptible. The AHAS protein from IMINTA 1 and IMINTA 4 was shown to be heat sensitive, with no detectable activity of AHAS after 1 min. of heating at 100 °C. AHAS from IMINTA 1 and IMINTA 4 were shown to be equivalent to parental controls with respect to trypsin degradation. The unmodified form of the AHAS protein shows no amino acid similarity to known allergens. The amino acid sequence of the mutated AHAS differs by one amino acid from that of unmodified rice.

BASF provided evidence to show that the protein components of IMINTA 1and IMINTA 4 are not altered in comparison with an unmodified comparator. High Performance Liquid Chromatography (HPLC) was run on protein extracts from unmodified and modified rice to indicate that no new major proteins or increased protein expression occurred as a result of the mutagenic event.

BASF has provided to the CFIA a method for the detection and identification of rice containing this novel trait.

3. Stable Expression

Herbicide treatments performed over several generations demonstrated that the herbicide tolerance trait was stably inherited and expressed in events IMINTA 1 and IMINTA 4.

IV. Criteria for the Environmental Assessment

Note: Events IMINTA 1 and IMINTA 4 will not be grown in Canada and will only be imported as human food or livestock feed. The majority of rice imported to Canada does not have an intact hull, which results in the seed being incapable of germination and growth. Due to the unfavorable climatic conditions for rice in Canada, it is unlikely that grain from events IMINTA 1 and IMINTA 4 would be capable of persisting in the Canadian environment.

1. Potential of events IMINTA 1 and IMINTA 4 to Become Weeds of Agriculture or Invasive of Natural Habitats

The centre of origin of rice is considered to be in the subtropics of Southeast Asia. Rice is not grown in Canada and is not adapted to the environmental conditions encountered in Canadian agricultural environments.

The CFIA evaluated data submitted by BASF on the biology of the events IMINTA 1 and IMINTA 4 and determined that vegetative vigour, time to maturity and seed production were within the normal range of expression of these traits currently displayed by commercial rice varieties.

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

Events IMINTA 1 and IMINTA 4 are not intended for cultivation in Canada and the novel trait has no intended effects on weediness or invasiveness. The CFIA has therefore concluded that these events have no altered weediness or invasiveness potential in Canada when compared to conventional rice varieties.

2. Potential for Gene Flow from events IMINTA 1 and IMINTA 4 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive

Species sexually compatible with rice do not occur in Canada. The wild "rice" which occurs in Canada (Zizania aquatica) belongs to a species that is not sexually compatible with domesticated rice (Oryza sativa). Events IMINTA 1 and IMINTA 4 will not be cultivated in Canada and if released, would not persist.

The CFIA has therefore determined that gene flow to sexually compatible species in Canada is not possible.

3. Altered Plant Pest Potential of events IMINTA 1 and IMINTA 4

Oryza sativa is not a plant pest in Canada, additionally the agronomic characteristics of events IMINTA 1 and IMINTA 4 were shown to be within the normal range of conventional rice varieties.

The CFIA has therefore determined that events IMINTA 1 and IMINTA 4 do not present a plant pest concern.

4. Potential Impact on Non-Target Organisms of events IMINTA 1 and IMINTA 4

The detailed characterization of the modified gene and the resulting enzyme, has led to the conclusion that the expression of the modified protein does not result in altered toxic or allergenic properties. The AHAS enzyme is not a known toxin, does not confer resistance to agricultural pests and is commonly found in nature with a history of safe use.

Based on the above, the CFIA has determined that events IMINTA 1 and IMINTA 4 will not result in altered impacts on non-target organisms, including humans, compared to current rice varieties.

5. Potential Impact on Biodiversity of events IMINTA 1 and IMINTA 4

These events are safe to non-target organisms, do not present altered weediness or plant pest potential and will not be grown in Canada. In addition, the novel trait has not altered the ability of these events to persist in the Canadian environment.

The CFIA has therefore concluded that events IMINTA 1 and IMINTA 4 will have no impact on biodiversity in Canada.

V. Nutritional Criteria Assessment as Livestock Feed

1. Potential Impact on Livestock Nutrition

Nutritional Composition and Anti-Nutritional Factors

Nutritional composition data was obtained from events IMINTA 1 and IMINTA 4 and the parental control IRGA 417, grown in 2 locations in Argentina. Grain samples were analyzed for protein, fibre, ash, branched-chain amino acids (valine, isoleucine and leucine), essential amino acids (cystine, methionine, threonine and lysine), fatty acids, minerals and vitamins. There were no statistically significant differences between the IMINTA events and IRGA 417 control for protein, fibre, ash, branched-chain and essential amino acids. Crude fat levels were significantly lower in the IMINTA events compared to the control, however the levels were comparable to conventional rice varieties. Oleic acid was significantly lower in the IMINTA events than IRGA 417, but was comparable to conventional rice varieties. Vitamins B1, niacin, B6, B2, folic acid and E were not statistically significantly different between the IMINTA events and IRGA 417. Pantothenic acid was significantly lower in the IMINTA events compared to the control, but was within the range of conventional rice varieties. There were no differences between the IMINTA events and the parental control for all minerals.

Phytic and trypsin inhibitor were analyzed in IMINTA1 and IMINTA 4 grain and compared to the parental control, IRGA 417. There were no statistically significant differences between the IMINTA events and IRGA 417 for phytic acid. Trypsin inhibitor was not detected for both the IMINTA events and the control.

The evidence provided by BASF supports the conclusion that the nutritional compositions of rice events IMINTA 1 and IMINTA 4 are equivalent to conventional rice varieties.

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

The AHAS enzyme is found in a wide variety of plants and micro-organisms. AHAS is not a known toxin or allergen and a single base pair change would not be expected to change this. AHAS from events IMINTA 1 and IMINTA 4 is feedback inhibited as is unmodified AHAS, it is present in small amounts in the feed, it is heat labile and it is rapidly degraded under conditions in the gastrointestinal tract. The expression of AHAS is not changed by the modification. Based on the information provided by BASF, the modified AHAS is unlikely to be a novel toxin or allergen.

Based on the detailed characterization provided (nutritional composition, agronomic data and HPLC protein profiles of the modified plant compared to the unmodified comparator) it is unlikely that secondary mutations causing unintended effects have occurred in the rice genome.

VI. New Information Requirements

If at any time, BASF becomes aware of any information regarding risk to the environment, including risk to human or animal health, that could result from release, in Canada or elsewhere, of rice events IMINTA 1 and IMINTA 4, their descendants, or products derived there from, BASF must immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of events IMINTA 1 and IMINTA 4 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 events IMINTA 1 and IMINTA 4.

VII. Regulatory Decision

Based on the review of data and information submitted by BASF, including comparisons of events IMINTA 1 and IMINTA 4 with unmodified rice counterparts, the Feed Section, CFIA, has concluded that the modified gene and its corresponding novel trait will not confer to events IMINTA 1 and IMINTA 4 any characteristic that would raise any concerns regarding safety or nutritional composition. Rice grain, groats, hulls, bran and rice bran oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. Events IMINTA 1 and IMINTA 4 have been assessed and found to be as safe as and as nutritious as traditional rice varieties. Events IMINTA 1 and IMINTA 4 and their products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada. These events will not be grown in Canada nor can the seed overwinter, therefore the release of the feed into the environment would result in neither intended nor unintended environmental effects.

Livestock feed use of IMINTA 1 and IMINTA 4 is therefore authorized as of October 30, 2006. Any rice lines derived from these events may be imported and/or released, provided no inter-specific crosses are performed, provided the intended use is similar, and provided it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown rice, in terms of their potential environmental impact and livestock feed safety.

Rice events IMINTA 1 and IMINTA 4 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 events IMINTA 1 and IMINTA 4.

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