Novel food information: Imidazolinone Herbicide Tolerant Rice RTC1

Novel food information

Health Canada has notified RiceTec Inc. that it has no objection to the food use of herbicide tolerant rice line RTC1. The Department conducted a comprehensive assessment of this rice line 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.

On this page

1. Background
   1. 1. Introduction
   2. 2. Development of the modified plant
   3. 3. Characterization of the modified plant
   4. 4. Product information
   5. 5. Dietary exposure
   6. 6. Nutrition
   7. 7. Chemistry
   8. 8. Toxicology
   9. 9. Allergenicity
2. Conclusion

Background

The following provides a summary of the notification from RiceTec Inc. and the evaluation by Health Canada. This document contains no confidential business information.

1. Introduction

RiceTec Inc. has developed a genetically modified (GM) rice line (Oryza sativa L.), RTC1, that exhibits tolerance to the acetohydroxyacid synthase (AHAS)-inhibiting herbicide imidazolinone (IMI). This novel trait is achieved through chemical mutagenesis and the successful introgression of a mutant AHAS enzyme into the rice genome by conventional breeding and selection.

The mutant AHAS enzyme in RTC1 possesses one amino acid substitution, A205V (alanine to valine at position 205) which confers herbicide tolerance, allowing the enzyme to function in the presence of IMI herbicides. Health Canada has previously assessed IMI-tolerant rice lines (CL121 and CL141) that had a different point mutation in AHAS (Health Canada, 2002). Health Canada has also previously assessed an IMI-tolerant sunflower line X81359 (Health Canada, 2003) possessing the same mutation in AHAS as RTC1.

Health Canada has previously indicated no objection to the sale of various IMI-tolerant crops (including Cibus Canola Line 5715, Health Canada, 2013; soybean BPS-CV127-9, Health Canada, 2012; Clearfield wheat variety Teal 11A, Health Canada, 2004; Clearfield lentil variety RH44, Health Canada, 2004; etc.).

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 reflects international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how herbicide tolerant rice RTC1 was developed, how the composition and nutritional safety of this variety compared to its unmodified comparator, and what the potential is for this variety to present a toxic or allergenic concern. RiceTec Inc. has provided data to support that this variety is safe for use as food in Canada.

The Food Directorate has a legislated responsibility for the 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). IMI-tolerant rice RTC1 is considered to be a novel food under the following part of the definition of novel foods:

1. "c) a food that is derived from a plant, animal, or microorganism that has been genetically modified such that
   1. iii. One or more characteristics of the plant, animal, or microorganism no longer fall within the anticipated range for that plant, animal, or microorganism".

2. Development of the modified plant

In rice, the first step in the branched-chain amino acid (i.e., valine, leucine, and isoleucine) biosynthesis pathway is catalysed by the AHAS enzyme, also referred to as acetolactate synthase (ALS). Imidazolinone (IMI) herbicides function by binding strongly to the active site of the AHAS enzyme, thereby inhibiting the branched-chain amino acid biosynthesis pathway which is essential for rice plant growth. IMI tolerance in rice lines is often attributed to point mutations in their ahas gene which prevents the binding of the herbicide to the AHAS enzyme.

IMI-tolerant rice RTC1 was produced through chemical mutagenesis of the parental variety P1003, using ethyl methanesulfonate (EMS). Mutagenized seeds were then grown and tolerant plants were selected using the herbicide imazethapyr. Tolerant plants were then backcrossed with the parental variety over six generations to fix the mutation within the parental background in the RTC1 variety.

3. Characterization of the modified plant

The mutation in the RTC1 rice line was characterised using Sanger DNA sequencing which confirmed a point mutation of C^®T (cytosine^®thymine) at nucleotide position 536 of the ahas gene. This DNA mutation results in a single amino acid substitution, alanine to valine, at position 205.

Segregation analysis was conducted to demonstrate the mode of trait inheritance of RTC1 and to confirm stability of the IMI-tolerance trait across multiple generations. Lines segregating for the mutation were systematically tested by the application of imazethapyr and then confirming trait inheritance using the allele-specific DNA markers. The expected 1:3 ratio of herbicide sensitivity to tolerance was observed for F2 populations. Inheritance patterns were also tracked by molecular marker analysis of backcrossed populations showing expected single gene segregation depending on the generation (1:1 ratio in F1 and 1:2:1 ratio in F2 populations). All Chi-square tests were found to be not significant, thus demonstrating stable Mendelian inheritance of the IMI-tolerance trait in RTC1.

A bacterially synthesized AHAS was produced in order to express adequate amounts of RTC1 AHAS for pepsin digestion as well as heat stability studies. GenScript Biotech used an expression system in Escherichia coli to produce the RTC1 AHAS enzyme. GenScript inserted a synthetic ahas gene (1965 bp) corresponding to the known RTC1 DNA sequence, encoding the RTC1 point mutation, into the vector pET-30a(+) and transformed the construct into E. coli BL21 (DE3). The synthetic RTC1 ahas gene sequence was optimized for bacterial codon usage and also encoded a C-terminal His tag for downstream purification of the expressed protein using a nickel column. The expressed RTC1 protein was purified further by dialysis and filter-sterilization using a 0.22 μm filter. SDS-PAGE and Western blot analysis, using an anti-His antibody, confirmed that the expressed RTC1 AHAS protein was a single polypeptide with the expected molecular mass (72.7 kDa).

The bacterially synthesized RTC1 AHAS protein was analysed using nano-LCMSMS with a Thermo LTQ Orbitrap mass spectrometer to confirm its amino acid sequence. This method confirmed 88% of the deduced amino acid sequence for RTC1 AHAS and the point mutation A205V was also confirmed via this method. The bacterially synthesized RTC1 AHAS was deemed to be equivalent to the RTC1 AHAS in rice by the following: amino acid sequence identity, including the point mutation of the synthetic AHAS, was confirmed via mass spectrometry; the correct molecular mass of the expressed synthetic protein as determined by SDS-PAGE and Western analysis; and the enzyme activity profile of the synthetic versus the plant-derived RTC1 AHAS were comparable.

Based on the information provided, there are no concerns regarding the food use of RTC1 rice from a molecular perspective.

4. Product information

Herbicide tolerant rice RTC1 differs from its conventional counterpart by the presence of a mutant AHAS-encoding gene in the host genome. Expression of this gene results in a mutant AHAS enzyme that is capable of functioning in the presence of AHAS-inhibiting herbicides, thus permitting herbicide tolerant rice RTC1 to grow in the presence of such herbicides, namely imidazolinones (IMIs).

RiceTec Inc. provided a rationale to address the potential changes to the expression of the IMI-tolerant AHAS enzyme in rice. It is expected that the point mutation in the IMI-tolerant RTC1 AHAS is highly unlikely to affect the expression levels of AHAS in RTC1 compared to wild-type AHAS expression in rice. Since the AHAS enzyme is critical for amino acid metabolism, its activity levels would be expected to be highly controlled. Any significant difference in expression of this enzyme would likely have an impact on the plant phenotype and be noted during the selection process. Based on the information provided, there are no concerns regarding the food use of RTC1 rice from a molecular perspective.

5. Dietary exposure

It is expected that herbicide tolerant rice RTC1 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 herbicide tolerant rice RTC1.

6. Nutrition

Compositional data for a hybrid containing the RTC1 genetic modification alone ("single mutant"), a hybrid of RTC1 with a line containing a previously assessed IMI-tolerant event ("double-mutant hybrid"), and a closely related, conventional hybrid ("control") were collected from three field trials conducted across two locations in Argentina during the 2015/2016 growing season. In each trial, three replicates of each entry were planted in a randomized complete block design. The single mutant and double-mutant hybrids were treated with IMI herbicide.

Grain samples were harvested and analyzed for proximates and fibres, fatty acids, amino acids, minerals, vitamins, and anti-nutrients (lectins, phytic acid, trypsin inhibitors). These compositional components are in line with recommendations listed in the Organisation for Economic Co-operation and Development (OECD) consensus document on compositional considerations for new varieties of rice. The analyses for each component were conducted using internationally approved and validated analytical methods.

The data were summarized as the mean, minimum, maximum, and standard deviation. Where a statistically significant difference (P-value < 0.05) between the modified and conventional hybrids was identified, further context for interpreting the nutritional relevance of the difference was obtained through comparison to the expected range for conventional rice as defined by means reported in the International Life Sciences Institute Crop Composition Database, the OECD consensus document, or the petitioner's submission based on compositional analysis of several conventional rice lines apart from the hybrid control.

Statistically significant differences between the single mutant and control were observed for the following components (control vs. single mutant, units): neutral detergent fibre (19.189 vs. 21.367, % DW), manganese (0.011 vs. 0.010, % DW), alpha-tocopherol (7.580 vs. 8.524, mg/kg DW), gamma-tocopherol (3.198 vs. 3.786, mg/kg DW). Since the modified cultivar was never outside the expected range for these components for conventional rice, these differences were not considered to be a nutritional safety concern.

Statistically significant differences between the double-mutant hybrid and control were observed for the following components (control vs. double-mutant hybrid, units): neutral detergent fibre (19.189 vs. 22.144, % DW), myristic acid (0.316 vs. 0.338, % total fatty acids). Since the modified cultivar was never outside the expected range for these components for conventional rice, these differences were not considered to be a nutritional safety concern.

The folate content of the double-mutant hybrid (3.123 mg/kg DW) was significantly higher than the control hybrid (1.717 mg/kg DW). It was noted that higher folate levels, compared to control, appeared to be restricted to only one of the two field locations. The petitioner provided supplemental data suggesting that higher levels of folate in the double-mutant hybrid may be attributed to differences in weed control and interspecific competition, as a result of comparing herbicide-treated to untreated hybrids, rather than to the genetic modification per se. Firstly, elevated folate levels were found when IMI-treated double-mutant hybrids were compared to untreated controls, but not when untreated double-mutant hybrids were compared to untreated controls. Secondly, elevated folate levels are not seen when IMI-treated double-mutant hybrids were compared to treated control hybrids carrying a genetic modification conferring IMI-tolerance. Furthermore, it was noted that the Tolerable Upper Intake Level (UL) for folate applies only to its synthetic form (i.e., folic acid) which is not naturally found in plants. Based on the current understanding of folate biosynthesis in plants, it seems extremely unlikely that the genetic modification inherent to the double-mutant hybrid would lead to accumulation of folic acid (versus natural forms of folate). Therefore, the observed difference in folate content was not considered to be a nutritional safety concern.

Based on the information provided, there were no safety concerns regarding the food use of RTC1 from a nutritional perspective.

7. Chemistry

Analytical data were not provided by the petitioner for lead, arsenic, cadmium or mercury. However, a compositional assessment was conducted comparing the single mutant hybrid (RTC1) and double-mutant hybrid (RTC1 line crossed with a line containing a previously-assessed IMI-tolerant event) and 3 control lines. Samples were assessed for levels of various nutrients, including the minerals calcium, copper, iron, magnesium, manganese, phosphorus, potassium, and zinc. As it was determined that levels of nutritive trace elements in RTC1 line and the double-mutant hybrid were comparable to the control lines, it is not expected that RTC1 rice line would uptake more toxic trace elements relative to non-modified rice.

Analytical data were not provided by the petitioner for mycotoxins. Mycotoxin susceptibility can result in reduced plant growth. However, the submission indicates that there are no differences observed in phenotypic data between the single-mutant (RTC1), double-mutant hybrid and their non-modified counterparts. This is taken as an indication that the genetic modification process did not give rise to unintended effects relating to mycotoxin susceptibility.

Based on the information provided, IMI-tolerant rice line RTC1 would not be expected to pose a concern to human health from a chemical contaminants perspective.

8. Toxicology

The toxicological evidence provided to demonstrate the safety of rice line RTC1 expressing the novel AHAS protein was evaluated. A safety rationale was provided by the petitioner which was based on the lack of systemic exposure or similarity to known toxins as demonstrated by in vitro assays and in silico analyses.

A heat lability assay demonstrated that the novel AHAS protein loses all enzyme activity when exposed to temperatures above 70 °C for 30 minutes (the earliest time point measured). A visual analysis by SDS-PAGE determined that heat exposure only partially degrades the protein. It was concluded that the novel AHAS protein loses its tertiary structure and enzymatic function at temperatures used to cook rice, but that it is not fully degraded under these conditions. The loss of enzyme activity suggests that the AHAS protein would not be active in cooked rice and would therefore not be capable of eliciting any toxic reactions.

A simulated gastric fluid (SGF) assay was performed to determine the stability of the novel AHAS protein in conditions similar to those in the human stomach. The protein was exposed to SGF for times ranging from 0.5 to 60 minutes. An SDS-PAGE analysis showed that the 70 kDa band representing the intact protein was no longer visible within 0.5 minutes, although a small 10 kDa fragment was observed. No protein bands were visible after 2 minutes. These results indicate that the novel protein is rapidly degraded under conditions similar to those found in the stomach and that no systemic exposure to the novel protein would be expected after the consumption of rice.

A search for amino acid sequence homologies between the mutated AHAS protein and known protein toxins did not find any meaningful matches. In all cases, the matches were either of low similarity (<35%), did not include the point mutation as part of the match, and/or were to hypothetical proteins.

Based on the available information, there were no toxicological concerns with the proposed use of rice line RTC1.

9. Allergenicity

The petitioner provided information and data to support the lack of allergenic potential for the novel AHAS protein. The petitioner conducted a search for amino acid sequence homologies between the mutated AHAS protein with known allergens. No matches with >35% homology over 80 amino acid residues with a probability of <1.0 were found. It was concluded that the novel AHAS protein does not resemble any known protein allergens listed in this database.

Additionally, the simulated gastric fluid assay described above demonstrated that it is unlikely that the intact protein would reach the intestine intact to elicit an allergic reaction.

The petitioner also provided a rationale to support the lack of changes to the levels of endogenous allergens in rice. According to the petitioner, the AllergenOnline database identifies several allergenic proteins in Oryza species belonging to the trypsin alpha-amylase inhibitor family. The petitioner argued that it is unlikely that the levels of these endogenous allergens would have been significantly altered or increased as a result of the mutagenesis procedure because the original mutant line was extensively back-crossed which would have removed the majority of the original mutated genetic material. To add to the rationale, the petitioner also demonstrated that the trypsin activity in line RTC1 was similar to that in conventional rice lines. Overall, there is no evidence to suggest that there is an increased risk of allergic reactions to endogenous proteins in rice line RTC1.

Based on the evidence presented, there are no allergenic concerns with respect to the proposed use of rice line RTC1.

Conclusion

Health Canada's review of the information presented in support of the use of imidazolinone (IMI)-tolerant rice variety RTC1 does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of RTC1 rice. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

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For further information, please contact:

Health Canada
Novel Food Section
Food Directorate
Health Products and Food Branch
251 Sir Frederick Banting Driveway
PL2204E
Ottawa, Ontario, K1A 0K9
hc.bmh-bdm.sc@canada.ca