Novel Food Information- Herbicide-Tolerant Rice – HPHI2 Event (Provisia®)

Health Canada has notified BASF that it has no objection to the food use of rice expressing endogenous acetyl-coenzyme A carboxylase (ACCase) containing a single amino acid mutation which confers tolerance to the aryloxyphenoxypropionate (FOP) and cyclohexanediones (DIM) group of herbicides used to control grass weeds. The Department conducted a comprehensive assessment of this rice event according to its Guidelines for the Safety Assessment of Novel Foods. These guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

The following provides a summary of the notification from BASF and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

ACCases are multifunctional enzymes that are ubiquitous in plants. Wild type rice contains two genes for ACCase, one that is active in the cytosol (OsACC1), which is intrinsically resistant to FOP and DIM herbicides, and one active in the plastid, a subcellular compartment of plant cells (OsACC2), which is sensitive to these herbicides. HPHI2 rice expresses a variant of OsACC2 containing a single amino acid change, I1781(Am)L (numbering is based on the plastidic ACC2ase protein of Alopecurus myosuroides, a grass species, abbreviated “Am” in this submission) within the substrate binding pocket in the carboxy terminal domain. This mutation confers resistance to DIM and FOP family herbicides. The mode of action for these herbicides is to block fatty acid biosynthesis, resulting in a loss of membrane integrity in growing cells and cell death. ACCase is regulated by feedback inhibition by an end-product of fatty acid biosynthesis, palmitoyl-CoA.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada’s Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents (e.g., Codex Alimentarius). The assessment considered: how HPHI2 rice (commercial name: Provisia®) was developed; how the composition and nutritional quality of HPHI2 rice compared to non-modified varieties; and what the potential is for HPHI2 rice to be toxic or cause allergic reactions. BASF has provided data that demonstrates that HPHI2 rice is as safe and of the same nutritional quality as traditional rice varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from HPHI2 rice are considered novel foods under the following part of the definition of novel foods:

“c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

(i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.  

2. Development of the modified plant

The petitioner provided information describing the methods used to develop HPHI2. Mature seeds from the Indica-1 line were dissected and grown in culture to produce clusters of somatic undifferentiated tissue, termed calli. The calli were propagated in culture and subject to four rounds of exposure to increasing concentrations of cycloxydim herbicide over several weeks. Tissue that grew in the presence of herbicide was induced to grow into plantlets. These were screened using polymerase chain reaction (PCR) methodology to detect the presence of spontaneous mutations within the OsACC2 gene. Plantlets containing the mutation of interest were regenerated into plants (termed the M0 generation). These were treated with cycloxidim herbicide at different concentrations, then exposed to drought conditions for one day, after which normal watering and substrate fertilizing was resumed. Plants were observed for sensitivity to herbicide over two weeks, and those with low or no levels of injury were retained and designated as the M1 generation. These were tested for growth characteristics and herbicide tolerance in the field. The HPHI2 line was selected and further characterized in laboratory and field studies, and then grown to produce subsequent generations. The M4 generation plants were used for all product safety studies.

3. Characterization of the modified plant

Molecular characterization of HPHI2 was accomplished by sequencing the OsACC2 gene in both the event and conventional Indica-1 rice. Plants of both varieties were grown in the greenhouse without the application of herbicide. Genomic DNA isolated from leaf tissue served as the template for PCR amplification. Seven overlapping PCR reactions were used to amplify the entire coding region of the gene, including non-coding regions 300 base pairs flanking the OsACC2 gene. Only one nucleotide difference was observed between the aligned contig sequences. This change would result in a deduced change in the expressed protein at position 1781 from isoleucine to leucine to give a mutated protein designated as OsACC2-I1781(Am)L. It was concluded that only one nucleotide change within OsACC2 differentiated rice event HPHI2 from its conventional counterpart, Indica-1.

A series of backcrossing and self-pollination steps were performed using traditional breeding methods in order to assess the stability of the introduced I1781(Am)L mutation. Both the genotype and the phenotype were determined for the progeny of each step, the former using a quantitative single nucleotide polymorphism (SNP) assay, and the latter by assessment of the herbicide tolerance trait. Taken together, the genotype and phenotype data gathered across hundreds of plants produced through different breeding generations was consistent with a model of inheritance of a single locus, OsACC2-I1781(Am)L, according to Mendelian principles. The inheritance of this trait, produced by a single nucleotide change, was stable throughout the breeding program.

4. Product Information

A reference protein was required as an antigen for generation of a primary antibody and as a standard both for quantitative western blot analysis and for digestion and stability studies.
Isolating sufficient quantities of the OsACC2-I1781(Am)L protein from Provisia® rice is difficult technically, and so a reference ACCase protein derived from native rice was expressed and purified in transgenic maize. The transgenic maize was produced by Agrobacterium-mediated transformation using a vector containing a cassette for overexpression of a double-mutant OsACC2 from rice. This version of OsACC2 was used because it was readily available and easy to purify due to the presence of a carboxy-terminal His-tagged. OsACC2-His contained two mutations: I à L at position 1781 and I à N at position 2041 (A. myosuroides numbering). The latter mutation, like I1781, is located within the active site cavity of the carboxy-terminal domain of OsACC2. The second mutation had been introduced with the intention of enhancing the tolerance toward ACCase inhibitor herbicides. Early testing of the plants bearing this mutation showed that their function was not altered.

A set of studies was conducted in order to determine whether OsACC2 expressed in native rice (Indica-1; control) and the purified maize-expressed double mutant His-OsACC2-I1781(Am)L/I2041(Am)N (reference) described were equivalent to OsACC2-I1781(Am)L in HPHI2 rice (test), and could therefore be used as valid standards for measuring the expression level of the protein in rice tissue and assessing protein stability. Rice extracts of HPHI2 and Indica-1 were enriched for OsACC2 by partial purification, and these were characterized by gel electrophoresis and rice tandem mass spectrometry with high coverage (87 % for Indica-1, 88 % for HPHI2, and 90 % for double mutant ACCase). Follow up studies of the peptide fragments generated in the mass spectrometry analysis demonstrated that all the regions relevant for antibody detection were characterized, and so the gaps in coverage did not raise concerns. The overall sequences and molecular weights of the three proteins were identical with the exceptions of the expected differences at residues 1781(Am) and 2041 (Am).

Anti-OsACC2 antibody reactivity was similar between the test (HPHI2 rice), control (Indica-1 rice), and reference (maize-expressed double mutant OsACC2) materials. Taken together, the protein characterization supports the claim that the maize-expressed reference protein can be used for measuring the expression level of the protein in rice tissue and assessing protein stability.

Wild type ACCase is subject to feedback inhibition whereby a metabolic product, palmitoyl-CoA, reduces enzymatic activity in a concentration dependent manner. Biochemical studies were performed in order to assess whether the I1781(Am)L mutation interferes with this regulatory function, and also whether the addition of herbicide impacts the enzymes activity in fatty acid biosynthesis. Assays were performed using protein extract preparations from the above-ground meristem portions of greenhouse-grown wild type (Indica-1, Wells) and mutant (HPHI2) plants. The functional characterization of wild type and mutant forms of OsACC2 showed that the single amino acid substitution in the OsACC2-I1781(Am)L expressed in HPHI2 results in the expected resistance to a FOP herbicide without altering the sensitivity to feedback inhibition by the endogenous metabolite palmitoyl-CoA.

The expression level and distribution of ACCase was measured in different rice plant tissues and at different growth stages in order to determine whether the I1781(Am)L substitution affected expression patterns. Both herbicide-treated and untreated plants were assessed. The His-tagged maize-expressed ACCase described above served as the reference material for the measurement of OsACC2 levels using quantitative western blot. Overall, it was found that the presence of the I1781(Am)L substitution did not alter the expression level or tissue distribution of ACCase during rice plant development. Likewise, the application of Targa® herbicide does not impact ACCase expression.

Based on the data presented regarding the protein characterization and expression, no concerns were raised regarding the food use of HPHI2.

5. Dietary Exposure

HPHI2 is the result of a genetic modification to confer tolerance to the FOP and DIM families of herbicides, with no intention to significantly alter nutritional parameters in the food. It is expected that HPHI2 will be used in applications similar to conventional rice varieties. The petitioner does not anticipate a significant change in the food use of rice with the introduction of HPHI2.

6. Nutrition

Rice samples for nutrient composition analysis were obtained from test (HPHI2) and control (Indica-1) plants grown in 2013 at five USA locations in a randomized complete block design with four plots cultivated with test, control, and reference varieties. All field trial experiments used to test HPHI2 rice were deemed acceptable. All analyses of test, control, and reference varieties were done using approved analytical methods and acceptable statistical methods.

Test, control, and reference varieties were analyzed for nutrients and anti-nutrients in grain, as follows: Proximate(s): protein, crude fat, ash, moisture, calculated carbohydrates, neutral detergent fiber (NDF), acid detergent fiber (ADF), total dietary fiber (TDF); Amino Acid(s): essential and non-essential; Fatty Acid(s): palmitic, stearic, oleic, linoleic, linolenic, arachidic, eicosenoic, lignoceric; Mineral(s): calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, zinc; Vitamin(s): folate, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, alpha-tocopherol, biotin; Anti-nutrient(s): phytic acid, lectins, trypsin inhibitors.

Across all 5 individual growing locations, statistical differences (HPHI2 treated with Targa® vs. control treated with standard herbicide) were noted in 11 analytes and only folate was statistically significant at two different sites in the same direction (lower). No other trends were noted in the data. For combined locations, a statistical difference was noted in one analyte, copper (1.08 mg/100g in HPHI2 treated with Targa vs 1.19 mg/100g in control, Indica-1). However, these levels for copper were both lower than the japonica reference varieties, and were similar to levels published in the OECD consensus document on key components of rice.

Based on the nutrient composition data presented, HPHI2 rice is similar to conventional commercial rice. No concerns were raised regarding the nutritional quality and safety of HPHI2.

7. Chemistry/Toxicology

A multistep approach was used to demonstrate the safety of event HPHI2 expressed in rice. This included: documentation of the history of use of the protein in genetically modified plants; characterization of the protein’s physicochemical and functional properties; quantification of protein expression levels in plant tissues; comparison of the protein coding sequences within the insert region to known allergens and toxins; evaluation of the proteins’ digestibility in simulated gastrointestinal and intestinal fluids (SGF and SIF); evaluation of protein stability to heat treatment typical of manufacturing of food ingredients and products; and an assessment of potential allergenicity of the expressed proteins.

The source organism, Oryza sativa L., has a long history of consumption as a staple food. Therefore, the use of a wild type Oryza sativa L. (Indica-1) as a parental line is not expected to pose a pathogenic or toxic concern.

ACCase enzyme has a well characterized, non-toxic mode of action. It is ubiquitous in plants and has a long history of safe consumption in food crops, including rice.

Bioinformatics analysis demonstrated that the OsACC2-I1781(Am)L protein does not have amino acid sequence similarity to known toxins, indicating this newly expressed protein is not expected to pose a toxic concern. 

Sethoxydim herbicide-tolerant corn contains ACCase with the same amino acid substitution as HPHI2 rice ACCase enzyme and was authorized by Health Canada in 1997.  Sethoxydim herbicide-tolerant corn has been commercialized for many years without any reported adverse health effects.

The level of ACCase protein in HPHI2 rice grain is relatively low (0.004 mg/g dry seed) and is the same as the levels in the parental and conventional rice varieties, indicating that the relatively low dietary exposure to this protein from HPHI2 rice is similar to the control rice varieties.

The OsACC2-I1781(Am)L protein was rapidly degraded in simulated gastric fluid and intestinal fluid. Therefore, even if it would survive under processing and cooking conditions, the OsACC2-I1781(Am)L protein would not be expected to be absorbed systemically and pose a health risk.

The OsACC2-I1781(Am)L protein was also rapidly inactivated by temperatures above 60 °C, indicating its lack of stability under processing and cooking conditions. Typically, rice is cooked at higher temperatures, and therefore no exposure to the active protein is anticipated. The denatured protein would be expected to be digested as any other dietary protein. As a result, the OsACC2-I1781(Am)L protein would not be expected to pose a health risk.

Based on these data and considerations, HPHI2 rice was assessed to be as safe as the conventional rice varieties from a toxicological perspective.

8. Allergenicity

The impact of the genetic modification in HPHI2 rice on the allergenicity of this rice as compared to conventional varieties was assessed. HPHI2 rice differs from wild type Indica-1 rice by a single base pair mutation in the coding region of OsACC2 gene. As mentioned above, this change is identical to that in sethoxydim herbicide tolerant corn, a line that has been commercialized for many years without any reported allergenic effects. The protein expression level in rice grain is low, and the mutated protein is rapidly degraded by digestive enzymes and inactivated by heat. Furthermore, bioinformatics analysis demonstrated that the OsACC2-I1781(Am)L protein does not show amino acid sequence similarity to known allergens. Based on these considerations, the modified protein would not pose an allergenic risk.

Rice is not a common allergenic food in Canada, however it does contain endogenous allergens. Based on the process used in this product’s development, which generated a single nucleotide change without the use of mutagens or recombinant DNA, it would not be expected to result in downstream effects generating an increase in the amount of endogenous rice allergens. It is considered unlikely that this product would contain more endogenous rice allergens than conventional rice varieties.

Based on the above, HPHI2 rice is not considered to pose an increased allergenicity concern as compared to the conventional rice varieties.

9. Conclusion

Health Canada’s review of the information presented in support of the food use of HPHI2 (Provisia®) rice does not raise concerns related to food safety. Health Canada is of the opinion that food derived from HPHI2 rice is as safe and nutritious as food from current commercial rice varieties.

Health Canada’s opinion deals only with the food use of HPHI2 rice. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the potential environmental and animal health issues associated with HPHI2 rice. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective with the commercial sale of products derived from Provisia® rice.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada.  This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

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For further information, please contact:

Novel Foods Section
Food Directorate                               
Health Products and Food Branch                
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca