Novel Food Information: Quizalofop-tolerant rice - RTA1

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Health Canada has notified RiceTec, Inc. that it has no objection to the food use of Quizalofop-tolerant rice variety RTA1, expressing endogenous acetyl-coenzyme A carboxylase (ACCase) containing a single amino acid mutation which confers tolerance to the aryloxyphenoxypropionate (FOP) herbicides used to control grass weeds. The Department conducted a comprehensive assessment of this rice variety 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 original notification from RiceTec, Inc. and the evaluation by Health Canada and contains no confidential business information.

1. Introduction

RTA1 Oryza sativa (rice) variety contains a genetic modification conferring tolerance to the herbicide Quizalofop (quizalofop-p-ethyl). This tolerance is the result of a single amino acid change in acetyl-CoA carboxylase (ACCase), an enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA in the synthesis of fatty acids. Herbicides such as quizalofop work by inhibiting the ACCase enzyme, thereby inhibiting fatty acid synthesis in plant tissue, resulting in a loss of membrane integrity in growing cells and cell death. RTA1 was developed through standard chemical mutagenesis, followed by conventional breeding. This rice line expresses a mutated form of the enzyme acetyl-coenzyme A carboxylase (ACCase), which confers tolerance to aryloxyphenoxypropionate (FOP) herbicides, such as quizalofop-p-ethyl (Quizalofop) used to control grass weeds.

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 in this area (e.g., Codex Alimentarius). The assessment considered: how this rice variety was developed; how the composition and nutritional quality of this variety compared to non-modified rice varieties; and the potential for this rice variety to be toxic or cause allergic reactions. RiceTec Inc. has provided data that demonstrate that RTA1 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 the Food and Drug Regulations(Division B.28). RTA1 is considered a novel food under the following part of the definition of novel foods:

2. Development of the Modified Plant

The petitioner has provided information describing the methods used to develop RTA1 and the molecular biology data that characterize the genetic change, which results in tolerance to Quizalofop.

The RTA1 rice was generated through standard chemical mutagenesis approach with sodium azide and methylnitrosurea, followed by conventional breeding. The selected herbicide tolerant line contains a single base change in the DNA sequence of the gene for ACCase that confers tolerance to quizalofop.

3. Characterization of the Modified Plant

Molecular characterization of RTA1 was accomplished by Sanger sequencing and PCR analysis of the mutated rice line and the parental line. The results showed one single base change between the mutant line and the parental line.

The genetic stability of the RTA1 trait was tested using a functional nucleotide polymorphic (FNP) DNA marker developed to detect the specific mutation. The phenotypic stability was established by assessing the herbicide tolerance trait through a Chi-square analysis. The genotype and phenotype data gathered across different breeding generations were consistent with Mendelian principles of inheritance. The results support the conclusion that the inheritance of this herbicide tolerant trait was stable throughout the breeding program.

To confirm that the single base change did not alter the expression of the ACCase gene RTA1, the mRNA expression level of the gene was compared to the native gene using Droplet Digital PCR (ddPCR). The analysis showed similar levels of expression of the ACCase gene in the wild type and the RTA1 mutant line. In addition, protein expression from the flour, roots, and shoots was compared between the wild type and the mutant line. The differences in ACCase activity between the RTA1 rice and wild type in these three rice tissues were not biologically significant.

4. Product Information

Given the low level of expression of ACCase in rice, it was not possible to extract and purify in sufficient quantity for in vitro stability studies. The common approach of cloning the plant gene into an E. coli vector for protein overexpression and purification using a polyhistidine tag, resulted in the large (2422 amino acid residue, 250 kDa) protein degrading into a mixture of fragments not suitable for biochemical analyses. A different strategy was then taken, wherein the C-terminal domain (672 amino acid residue), containing the single mutation of interest, was cloned into the E. coli-based overexpression system, described above. The protein domain was purified under denaturing conditions and renatured. Mouse monoclonal antibodies were raised against this C-terminal domain, and were shown in Western blot analysis to detect both wildtype and mutant full-length ACCase expressed in plant, supporting the overall preservation of the domain structure. Equivalence of the rice-produced ACCase and E. coli-produced protein was further demonstrated by comparison of their molecular weights via both SDS-PAGE and mass spectrometry, and amino acid sequences through LC-MS/MS. Taken together, the results of these analyses support the use of E. coli-produced ACCase protein to evaluate the safety of the plant-produced protein.

5. Dietary Exposure

RTA1 rice is the result of a genetic modification to confer tolerance to the FOP family of herbicides, with no intention to significantly alter nutritional parameters in the food. It is expected that RTA1 rice 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 RTA1.

6. Nutrition

Compositional data for V3501-RTA1 (test), V3501 (control), and two reference varieties collected from four field trials in Argentina during the 2017-2018 growing season was provided. The test line was both treated and un-treated with quizalofop. In each trial, four replicates were planted in a randomized complete block design. Typical commercial agriculture production practices were used for the field trials.

Grain samples were harvested and analysed for moisture, dry matter, crude protein, crude fat, acid detergent fibre, total dietary fibre, neutral detergent fibre, ash, carbohydrates, amylose, calcium, copper, iron, magnesium, manganese, phosphorous, potassium, zinc, riboflavin, thiamine, niacin, pantothenic acid, vitamin B6, folic acid, biotin, tocopherols, amino acids, fatty acids, phytic acid, trypsin inhibitor, and lectin. The data provided was for all key nutrients and anti-nutrients as described in the Organization for Economic Co-Operation and Development (OECD) "Revised Consensus Document on Compositional Considerations for New Varieties of Rice (Oryza sativa): Key Food and Feed Nutrients, Antinutrients and Other Constituents" (2016).

In the data set provided statistically significant differences were observed in crude protein, carbohydrates, amylose, copper, manganese, phosphorous, potassium, zinc, glutamic acid, proline, serine, methionine, cysteine, lysine, tryptophan, threonine, phenylalanine, glycine, riboflavin, thiamine, niacin, vitamin B6, gamma-tocopherol, and oleic, arachidic, eicosenoic, behenic, docosadienoic, and lignoceric acids. In all cases the differences were small, and the analyte levels were within the reference ranges provided by the petitioner and the expected range for conventional rice. These differences were not considered nutritionally relevant. There were no differences in anti-nutrient content between the two lines.

Biotin was more than double in the untreated V3501-RTA1 line (18 μg/100 g) compared to the control (8 μg/100 g) and the mean falls outside the expected range. Biotin has defined adequate intake (AI) values based on life stage group (e.g., 30 μg in adults) but no tolerable upper intake level (UL) for biotin has been established as no adverse effects of biotin consumption in humans or animals have been reported. In addition, biotin is widely distributed in natural foodstuffs at varying concentrations (e.g., 100 μg/100 g in liver to 1 μg/100 g in fruits). Since biotin has no UL and the amount present in V3501-RTA1 is well within the range commonly found in foods, it would not present a safety concern.

There was no difference in total crude fat and rice is not a major nutritional source of fat; however, there were some statistically significant differences in fatty acid levels between V3501-RTA1 and control. Palmitic and linolenic acid are slightly higher and stearic acid is slightly lower in V3501-RTA1 compared to control. These three fatty acids each differed from control by less than 1, as a percentage of total fatty acids. For palmitic acid, both the control and V3501-RTA1 had a mean level that was outside the expected range. A higher palmitic acid level compared to commercial reference varieties may be inherent to the V3501 line. Changes in V3501-RTA1 to palmitic (increasing by 0.8) and stearic acid (decreasing by 0.9), both of which are saturated fatty acids, are expected to have a minimal impact on total saturated fatty acid intake. Linolenic acid is an essential omega-3 fatty acid that synthesizes long chain polyunsaturated fatty acids (e.g. EPA and DHA), although at a low efficiency. Linolenic acid is a very minor fatty acid in rice, and considering the low fat content of rice, the overall impact of the difference in V3501-RTA1 vs control rice would be negligible, in terms of linolenic acid intake.

Based on the information provided, no nutritional safety concerns were identified with the use of RTA1 rice.

7. Chemistry/Toxicology

A compositional assessment of RTA1 rice was presented, in which the concentrations of eight mineral nutrients were found to be similar to those of conventional controls and reference varieties. In cases where statistically significant differences were observed, the differences were small in magnitude, and not biologically significant.

Four field trials conducted in 2017-18 in Argentina showed that a variety of agronomic characteristics related to growth, plant health, and yield of RTA1 rice were comparable to those of conventional controls and reference varieties. This information suggests that the RTA1 rice is not expected to exhibit an increased uptake of toxic trace elements or susceptibility to infection by mycotoxigenic fungi relative to conventional rice. Furthermore, while mycotoxins have been occasionally observed at low levels in rice and rice products, these have not been identified as significant contributors to total dietary exposure to these compounds.

Based on the above information, RTA1 rice would not be expected to pose a concern to human health from a chemical contaminants perspective.

The modification in RTA1 rice is the induction of a single point mutation, resulting only in a single amino acid substitution in its ACCase enzyme. The native ACCase enzyme is well characterized, ubiquitously present in all plant species, and has a long history of safe consumption with no intrinsic toxic activity. Herbicide-tolerant novel foods carrying a similar trait (i.e., a single amino acid modification in ACCase) are already approved in Canada and have not been associated with any adverse effects (Corn lines DK412SR and DK404SR, 1997; and Provisia rice, 2016).

ACCase is not a major rice protein, and is primarily found in actively growing portions of the plant (e.g., leaf, shoots, roots). As such, dietary exposures to this protein from rice grains is expected to be low. The petitioner quantified the expression of the modified ACCase in RTA1, and found it was not increased relative to the native ACCase, therefore the dietary exposures to this enzyme will not be increased compared to the non-modified form from conventional rice. In addition, the petitioners demonstrated that the functional activity of the modified ACCase in RTA1 is not significantly altered relative to the native ACCase. Thus, given that there are no major alterations in the sequence of RTA1 ACCase, and the structure (and resultant expression and activity) are similar to that of the native enzyme, it is unlikely that the single amino acid modification will result in the development of a novel toxic mode of action.

Bioinformatics analysis demonstrated that the RTA1 ACCase protein does not have amino acid sequence similarity to any known toxins. The RTA1 ACCase was rapidly degraded in simulated gastric fluid, indicating that if any protein remained after processing and cooking conditions, the protein would be rapidly degraded in the acidic conditions of the stomach, and would not be absorbed systemically to pose a health risk.

Based on these data and considerations, RTA1 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 RTA1 rice on the allergenicity of this rice as compared to conventional rice varieties was assessed. ACCase enzymes are naturally present in rice, and this class of enzymes is not classified as an allergen. The petitioner conducted an amino acid sequence homology search to identify potential cross-reactivity of the modified ACCase in RTA1 against known allergens, and did not identify any matches against known allergens. Furthermore, the RTA1 ACCase protein is rapidly degraded by digestive enzymes. Based on these considerations, the modified protein would not pose an allergenic risk.

Rice (Oryza sativa) is not a common allergenic food and is not listed as a priority food allergen in Canada. Although rare cases of allergenic reactions in sensitized individuals have been associated with certain rice proteins, there is no expectation that a single point mutation in the structural domain of the ACCase gene would affect the expression of other rice proteins.

Based on the above information, RTA1 rice is not considered to pose an increased allergenicity concern as compared to conventional rice varieties.


Health Canada's review of the information presented in support of the food use of RTA1 rice does not raise concerns related to food safety. Health Canada is of the opinion that food derived from RTA1 rice is as safe and nutritious as food from current commercial rice varieties.

Health Canada's opinion deals only with the food use of RTA1 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 RTA1 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 RTA1 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.

(Également disponible en français)

For further information, please contact:

Health Canada
Novel Food Section
Food Directorate
Health Products and Food Branch
251 Sir Frederick Banting Driveway
Ottawa, Ontario, K1A 0K9

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