Novel Food Information: ROXY® rice expressing an oxyfluorfen herbicide tolerance characteristic

On this page

• Background
  1. Introduction
  2. Development of the Modified Plant
  3. Characterization of the Modified Plant
  4. Product Information
  5. Dietary Exposure
  6. Nutrition
  7. Chemistry
  8. Toxicology
  9. Allergenicity
• Conclusion

Background:

Health Canada has notified the California Cooperative Rice Research Foundation (CCRRF) that it has no objection to the food use of ROXY^® rice. 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 characteristics.

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

1. Introduction

The CCRRF has developed a novel rice (Oryza sativa L.) line, ROXY^® rice that exhibits tolerance to oxyfluorfen herbicide.

This novel characteristic was achieved through chemical mutagenesis and conventional breeding methods resulting in a single nucleotide deletion of guanine in Exon 8 at position 1699 in the UGP3 gene (i.e., referred to as the ROX1.1 mutation). This deletion causes a frameshift that results in truncation at amino acid 584 in the ROXY^® rice Uridine diphosphate (UDP)-Glucose Pyrophosphorylase 3 (UGP3) enzyme.

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 ROXY^® rice 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. The CCRRF 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). At the time the submission was made, foods derived from ROXY^® rice were considered to be a novel foods under the following part of the definition of novel food: “c) a food that is derived from a plant, animal, or microorganism that has been genetically modified such that

1. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.”

As of May 2022, under the newly published Guidance on the Novelty Interpretation of Products of Plant Breeding, foods derived from this rice variety are not considered to be novel foods.
However, as this product was considered novel at the time of submission (September 2021), the CCRRF requested that Health Canada complete the pre-market assessment.

2. Development of the Modified Plant

ROXY^® rice was produced through chemical mutagenesis of the parental variety M-206, using ethyl methanesulfonate (EMS). Mutagenized seeds were then grown and tolerant plants were selected using oxyfluorfen herbicides (CAS No. 42874-03-3). A total of nine mutant lines showed consistent oxyfluorfen tolerance from which a single mutant line was crossed back with the parental variety M-206, selected and advanced to produce the 17Y3000 line (containing the ROX1.1 mutation) that was used for assessment.

No antibiotic resistance marker genes, or other marker genes, were used in the development of ROXY^® rice.

3. Characterization of the Modified Plant

Genomic detection of the ROX1.1 mutation at position 1699 of the UGP3 gene was conducted using SNP ROX1.1 markers by standard PCR reaction and gel electrophoresis.

Segregation analysis was conducted for herbicide tolerance in the F3 generation of the 17Y3000 line to analyze the inheritance pattern of the ROX1.1 mutation. Data from the analysis demonstrated that the mutation is inherited according to Mendelian principles. Furthermore, genotype data over four generations of the 17Y3000 line confirmed that the ROX1.1 mutation is stably inherited while observations showed stable expression of the oxyfluorfen tolerant characteristic.

A recombinant UDP3 containing the ROX1.1 mutation was produced in order to express adequate amounts of mutant protein for digestion and thermolability studies. The Bureau of Microbial Hazards (BMH) confirmed the recombinant mutant UGP3 to be equivalent to the rice-produced mutant UGP3 based on the verified DNA sequences, the molecular mass of the expressed recombinant protein as determined by SDS-PAGE, and immunological analysis.

Based on the available data provided, the BMH has no safety concerns regarding ROXY^® rice from a molecular perspective.

4. Product Information

ROXY^® rice differs from its conventional counterpart by the presence of the mutant UGP3- encoding gene in the host genome.

UGP3 is a critical enzyme in the sulfolipid biosynthesis pathway. It is involved in the generation of UDP-glucose and is the committed enzyme for the first step of sulfolipid biosynthesis. The polypeptide of UGP3 contains a putative pyrophosphorylase consensus motif and a nucleotide- binding motif. These structural features of the protein sequence suggest that UGP3 is a chloroplast-localized UGP for the generation of UDP-glucose from glucose-1-phosphate and UTP. A comparative genomic study of UGP3 homologs across different plant species, including rice, suggests structural and functional conservation of these proteins, and thus, a committed role for UGP3 in sulfolipid biosynthesis.

It is hypothesized that the truncation in the UGP3 enzyme affects assembly of the sulfolipid polar heads in the cell membrane thereby altering the binding sites of the herbicide to the plant. Since the recognition site of the herbicide is altered, the ensuing peroxidation events and cell membrane destruction events cannot proceed, resulting in the modified plant’s tolerance to the herbicide.

Analysis of the modified UGP3 gene in the 17Y3000 line reveals a single nucleotide deletion that results in a frameshift and truncation of the ROXY^® rice UGP3 protein at position 584. There is no indication that this change would result in any change to the expression level of the gene or the amount of the enzyme present in the plant. To confirm this, crude protein extracts of both the mutant-expressing 17Y3000 line and the parental line, M-206, were made.

With no dilution, the amount of UGP3 detected in 17Y3000 leaf tissue crude protein extract samples was 11.2 ng/mL and the amount of UGP3 detected in M-206 leaf tissue samples was 2.6 ng/mL.

Grain extracts from the 17Y3000 and M-206 lines were analyzed for detection of UGP3 using semi-quantitative immunoblot techniques UGP3 was not detected in any grain extract sample from the mutant, oxyfluorfen-tolerant rice line 17Y3000, nor the wild type parental line M-206. Though UGP3 protein was not detected in the grain samples using the Bradford assay, coomassie brilliant blue (CBB) stain showed that grain protein was present in the gel.

The lack of detection of UGP3 protein in 17Y3000 and M-206 lines using protein immunoblots indicates that UGP3 is a low abundance protein in rice.

5. Dietary Exposure

It is expected that ROXY^® 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 ROXY^® rice.

6. Nutrition

The petitioner provided compositional data for experimental rice line 17Y3000 (i.e., ROXY^® rice), and parental isoline M-206 (control) grown at four locations in California during the 2019 growing season. Cultivated rice is not grown in Canada due to unsuitable temperatures and climate conditions; therefore, US field trial data was considered acceptable. In each field trial, three replicates of each entry (i.e., 17Y3000 and control) were planted in a randomized complete block design.

Grain samples were harvested and analyzed using acceptable methods for proximates (ash, crude fat, crude protein, moisture, acid detergent fiber, neutral detergent fiber, total dietary fiber); minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium and zinc); vitamins (thiamin, riboflavin, niacin, pantothenic acid, pyridoxine, biotin, folic acid and alpha tocopherol); phytic acid, trypsin inhibitor, lectins, amino acids; and fatty acids. The data were provided for all key components (nutrients and anti-nutrients) as described in the Organisation for Economic Co-operation and Development (OECD) Consensus Document on Rice (2016)^{Footnote 1}.

Statistically significant (P < 0.05) differences between 17Y3000 and control were noted in the compositional data provided. Specifically, differences in the mean values were observed for moisture, neutral detergent fiber, thiamin, riboflavin, and zinc. All values were within the reference ranges described in the OECD consensus document on rice (2016) and therefore were not considered to be nutritionally significant. No statistically significant differences between 17Y3000 and control were noted for crude protein or individual amino acids.

Rice is not a good source of fat (< 3 %) therefore, as expected, the majority of the fatty acids were below the level of quantification. The results of total fat and individual fatty acids showed the same pattern as the other analytes, where some statistically significant differences between 17Y3000 and control were noted, but these differences in the mean values were not considered nutritionally relevant.

Location effects were observed for proximates, amino acids, vitamins, fatty acids and some minerals (calcium, copper, iron, magnesium, manganese, phosphorus, and zinc). However, this was not considered a nutritional concern since environment and soil conditions are known to affect composition.

Based on the data provided by the petitioner, the Bureau of Nutritional Sciences (BNS) has not identified any nutritional concerns related to the proposed use of ROXY^® rice in the Canadian market place.

7. Chemistry

Chemical contaminant residue data was not provided by the CCRRF, nor have any unique contaminant considerations been identified with respect to ROXY^® rice. All types of rice sold in Canada must comply with Health Canada’s maximum levels (ML) set out in the List of Contaminants and Other Adulterating Substances of 0.2 ppm inorganic arsenic in polished (white) rice and 0.35 ppm in husked (brown) rice, with inorganic arsenic calculated as the sum of arsenite (As III) and arsenate (As V). As well, Health Canada has established a ML of 0.1 ppm inorganic arsenic for rice-based foods intended specifically for infants and young children set out in the above List.

As with any food or food ingredient sold in Canada, it is the responsibility of the food manufacturer to ensure that its use does not result in a violation of Section 4(1)(a) and (d) of the Food and Drugs Act, which states that no person shall sell an article of food that has in or on it any poisonous or harmful substance or is adulterated. If an elevated concentration of any chemical contaminant is found in any type of food, the Bureau of Chemical Safety (BCS) may conduct a human health risk assessment to determine if there is a potential safety concern and whether risk management measures are required.

8. Toxicology

The only modification in ROXY^® rice is the introduction of a single point mutation (deletion of guanine in exon 8 at position 1699), resulting in truncation of the UGP3 protein from 866 amino acid in the wild type to 584 amino acid in the notified rice. The native UGP3 protein is well characterized, commonly present in plant species including rice, and has a long history of safe consumption with no intrinsic toxic activity.

UGP3 is not a major rice protein and as such, dietary exposures to this protein from rice grains is expected to be low. An ELISA-based protein expression assay conducted by the petitioner demonstrated that the expression of the modified UGP3 from the grain of ROXY^® rice is equivalent to the native UGP3 from the grain of wild-type rice. Thus, there is no expectation that dietary exposures to this protein will be increased compared to the non-modified form from conventional rice.

The petitioner conducted an in silico search of the National Center for Biotechnology Information (NCBI) protein database to compare the amino acid sequence of the modified UGP3 in ROXY^® rice to known toxins. The results were analyzed for the keywords “toxin” or “toxic”. The search did not identify any noteworthy similarities to protein sequences of known toxins.

A heat stability assay conducted by the petitioner demonstrated that the modified UGP3 is susceptible to heat degradation under conditions relevant for preparation of rice (i.e., boiling [100°C] of the modified UGP3 protein for up to 30 minutes resulted in degradation of approximately 62% of the protein, while approximately 38% remained intact/stable).

Additionally, in an in vitro digestion assay conducted by the petitioner, the modified UGP3 was demonstrated to be rapidly degraded in simulated gastric fluid (SGF). Thus, even if the (low levels of) protein remain stable throughout the processing and cooking conditions of rice, the protein would be susceptible to rapid digestion in acidic conditions of the mammalian stomach, and would not be absorbed systemically to pose a health risk.

In considering the above, the overall weight-of-evidence suggests that ROXY^® rice is as safe as conventional rice from a toxicological perspective.

Based on the available toxicity data, the Bureau of Chemical Safety (BCS) has not identified any toxicological food safety concerns with use of ROXY^® rice as proposed.

9. Allergenicity

UGP3 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 using the AllergenOnline database^{Footnote 2} to identify potential cross-reactivity of the modified UGP3 in ROXY^® rice against known allergens. Searches included a full FASTA alignment, a sliding window of 80 amino acids stretches with greater than 35% identity, and a search for 8 contiguous amino acids to assess for presence of epitopes^{Footnote 3} (Codex, 2009^{Footnote 4}). The search did not identify any matches against known allergens.

Furthermore, as noted previously, the UGP3 protein in ROXY^® rice is rapidly degraded in SGF and therefore, it is expected that this protein or its resultant peptides would not be absorbed intact to pose an allergenic risk.

Rice 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 UGP3 gene would affect the expression of other rice proteins.

In considering the above, the overall weight-of-evidence suggests that ROXY^® rice does not pose any additional allergenic risk in comparison to conventional rice.

Based on the information provided, the Bureau of Chemical Safety (BCS) has not identified any allergenic concerns with the use of ROXY^® rice as proposed.

Conclusion:

Health Canada’s review of the information presented in support of the use of ROXY^® rice does not raise concerns related to food safety.

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

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:

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