Decision document DD2019-124: Determination of the safety of RiceTec Inc.'s rice (Oryza sativa L.) event RTC1

This decision document has been prepared to explain the regulatory decisions reached under Section 2.6 – Guidelines for the assessment of novel feeds: Plant sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards, and based on the environmental criteria in Directive 94-08 – Assessment criteria for determining environmental safety of plants with novel traits.

The Canadian Food Inspection Agency (CFIA), specifically the Animal Feed Program (AFP) of the Animal Health Directorate, with input from the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has evaluated information submitted by RiceTec Inc. This information concerns the imazethapyr tolerant rice event RTC1. The CFIA has determined that feed derived from rice event RTC1 does not present a significant risk to the environment, nor does it present livestock feed safety or nutrition concerns when compared to currently commercialized rice varieties in Canada.

Taking into account this evaluation, use as livestock feed of rice event RTC1, is therefore, authorized by the Animal Feed Program of the Animal Health Directorate as of June 4, 2019. Any rice lines derived from rice event RTC1 may also be used as livestock feed, provided that

• no inter-specific crosses are performed
• the intended uses are similar
• it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently commercialized rice varieties and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety and nutrition
• the novel genes are expressed at a level similar to that of the authorized line

Rice event RTC1 is subject to the same phytosanitary import requirements as unmodified rice varieties. Rice event RTC1 is required to meet the requirements of other jurisdictions, including but not limited to, the Food and Drugs Act and the Pest Control Products Act.

Please note that the livestock feed assessment of novel feeds is a critical step in the potential commercialization of these plant types. Other requirements, such as the assessment of novel foods by Health Canada, have been addressed separately from this review.

June 4, 2019

This directive was created by the Canadian Food Inspection Agency. For further information, please contact the Canadian Food Inspection Agency.

On this page

• 1. Brief identification of the modified plant
• 2. Background information
• 3. Description of the novel trait
  • Development method
  • Tolerance to imazethaphyr
  • Stable expression
• 4. Criteria for the environmental assessment
  • Potential of rice event RTC1 to become a weed of agriculture or be invasive of natural habitats
  • Potential for gene flow from rice event RTC1 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
  • Potential for rice event RTC1 to become a plant pest
  • Potential impact of rice event RTC1 and its gene products on non-target organisms, including humans
  • 5. Potential impact of rice event RTC1 on biodiversity
• Criteria for the livestock feed assessment
  • Potential impact of rice event RTC1 on livestock nutrition
  • Potential impact of rice event RTC1 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed
• 6. New information requirements
• 7. Regulatory decision

1. Brief identification of the modified plant

Designation of the modified plant: Rice event RTC1

Applicant: RiceTec Inc.

Plant species: Rice (Oryza sativa L.)

Novel traits: Tolerance to imazethapyr (imidazolinone class of herbicides)

Trait introduction method: mutagenesis and conventional breeding

Intended use of the modified plant: Production of rice for livestock feed, human food and industrial uses. Rice event RTC1 will be used in the same manner as conventional rice as livestock feed. This new rice event will be grown outside Canada, in the usual production areas for rice. Rice grain, rice hulls, and rice bran with germ will be imported into Canada for livestock feed use only.

2. Background information

RiceTec Inc. has developed a rice event that is tolerant to imazethapyr, an imidazolinone herbicide. Rice event RTC1 was developed by RiceTec Inc. using chemical-induced mutagenesis and conventional breeding to introduce a single nucleotide mutation in the acetohydoxyacid synthase (AHAS) gene, resulting in a single amino acid substitution, leading to the expression of the modified AHAS protein. The resultant amino acid substitution within the endogenous rice AHAS enzyme prevents binding of imidazolinone class of herbicides (for example, imazethapyr) to the AHAS protein, and therefore confers tolerance to the herbicide.

RiceTec Inc. has provided information on the identity of rice event RTC1, a detailed description of the modification method, breeding history, and levels of protein expression in the plant. The modified AHAS protein in rice event RTC1 was identified and characterized. Information was provided for the evaluation of the potential toxicity of the modified AHAS protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans through bystander exposure and to livestock.

Rice event RTC1 was field tested at 2 locations in Argentina (2015/2016) and 1 location in Brazil. An unmodified control rice variety, which shares the same genetic background as rice event RTC1, was included in the trials to act as a comparator for rice event RTC1. Two commercial herbicide tolerant inbred lines were also included in the field trials to establish ranges of comparative values that are representative of currently grown rice varieties.

Agronomic and phenotypic characteristics of rice event RTC1, such as stand count, seedling vigour, days to heading, days to maturity, plant height, grain length, width and yield were compared to those of the unmodified control rice variety, to a hybrid control containing a commercially approved IMI tolerant line and to the range established by the reference rice varieties.

Nutritional components of rice event RTC1 grain, such as protein, fat, moisture, ash, fibre, amino acids, fatty acids, vitamins, minerals and anti-nutrients were compared to those of the unmodified control rice variety and to a hybrid control containing a commercially approved IMI tolerant line. Values were compared to ranges established by the conventional rice varieties.

The Animal Feed Program (AFP) of the Animal Health Directorate, CFIA, with input from the Plant and Biotechnology Risk Assessment Unit of the Plant Health Science Directorate, CFIA, has reviewed the above information. The following assessment criteria, as described in Section 2.6 Guidelines for the Assessment of Novel Feeds: Plant Sources, of Chapter 2 of the RG-1 Regulatory Guidance: Feed Registration Procedures and Labelling Standards and Directive 94-08 – Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits were used to determine the safety and efficacy as livestock feed and the environmental safety of this novel feed:

• the potential impact of rice event RTC1 on livestock nutrition
• the potential impact of rice event RTC1 on animal health and human safety, as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed
• the potential for rice event RTC1 to become a weed of agriculture or to be invasive of natural habitats
• the potential for gene flow from rice event RTC1 to sexually compatible plants whose hybrid offspring may become more weedy or more invasive
• the potential for rice event RTC1 to become a plant pest
• the potential impact of rice event RTC1 and its gene products on non-target organisms, including humans
• the potential impact of rice event RTC1 on biodiversity

The AFP has also considered whether feeds derived from rice event RTC1 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

3. Description of the novel trait

Development method

Rice event RTC1 was developed by RiceTec Inc. using chemical-induced mutagenesis and conventional breeding. Seeds were treated with ethylmethylsulfonate (EMS) generating a bulk population. Plants were screened in the field for imazethaphyr tolerance. Resulting survivors were selected based on their tolerance to the imazethaphyr herbicide and were exposed to another round of selection. In order to fix the mutations conferring the herbicide tolerance, selected plants were converted to homozygous inbred lines through further breeding and selection. Mutations in tolerant rice lines were identified by means of targeted DNA sequencing and amino acid sequences prediction. These mutations were identified as a single nucleotide mutation which resulted in a single amino acid substitution in the AHAS protein. Rice event RTC1 was identified as a successful mutant based on molecular analyses and agronomic evaluations and was chosen for further development.

Tolerance to imazethaphyr

Imidazolinone herbicides act by disrupting the function of the acetolactate synthase (ALS) also referred as acetohydroxyacid synthase (AHAS). This enzyme is involved in the biosynthesis of branched chain amino acids (that is, valine, leucine and isoleucine) and its inhibition results in a lethal decrease in protein synthesis.

Rice event RTC1 was generated through chemical-induced mutagenesis with ethylmethylsulfonate and selected based on their tolerance to imazethapyr. Tolerant lines were analysed by targeted DNA sequencing. Protein sequence prediction revealed that a single base change in the DNA sequence of the AHAS gene caused the single amino acid substitution of Alanine to Valine.

AHAS protein expression in rice event RTC1 is driven by a constitutive promoter. The AHAS expression in rice event RTC1 was determined by ribonucleic acid (RNA) expression levels and by enzymatic activity. The RNA expression levels in different tissues (leaf, root, seed and whole plant) in rice event RTC1 were compared to the AHAS expression levels in an RTC1 hybrid and an unmodified hybrid control. Differences observed in the RNA expression levels between rice event RTC1, the RTC1 hybrid and the unmodified hybrid control were not considered significant.

The AHAS activity assay demonstrated that enzymatic activity between the AHAS expressed in rice event RTC1 (homozygous inbred line) and the AHAS expressed in the unmodified control, across a range of tissues (shoot, root, flour and germinating seed) was not significantly different.

To perform the safety studies on the modified AHAS protein, sufficient quantities of protein were obtained by expressing the AHAS mutated protein in an Escherichia coli (E. coli) production system. Equivalency between the rice event RTC1 expressed AHAS protein and an E. coli-expressed AHAS protein was demonstrated by comparing their amino acid sequence, enzymatic activity and heat stability. The apparent molecular size of the bacterially expressed AHAS was confirmed by SDS-PAGE. The amino acid sequence of the E. coli-purified protein was further analysed by peptide mass fingerprinting using mass spectrometry. Based on these results, the E. coli-expressed AHAS protein was found to be equivalent to the rice event RTC1 expressed AHAS protein.

The potential allergenicity and toxicity of the modified AHAS protein to livestock were evaluated. The source of the AHAS gene, rice (Oryza sativa L.), is not known to produce allergens and the AHAS protein amino acid sequence lacks relevant similarities to known allergens and toxins. Unlike many allergens, the AHAS protein is expressed at low levels in rice event RTC1. Moreover, the protein is rapidly degraded in simulated gastric and is heat labile, thus reducing the potential livestock exposure. The weight-of-evidence approach taken indicates that the modified AHAS protein is unlikely to be allergenic or toxin.

For a more detailed discussion of the potential allergenicity and toxicity of the modified AHAS protein, see Section 5: Potential impact of rice event RTC1 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed.

Stable expression

The single nucleotide mutation in the AHAS gene resulting in the herbicide-tolerance trait in rice event RTC1 was shown to be inherited according to Mendelian principles across multiple generations. The inheritance and stable expression of the herbicide-tolerance trait was verified by genotypic and phenotypic assays over 9 generations. The single nucleotide polymorphism (SNP) assay based on quantitative polymerase chain reaction (qPCR) confirmed the presence of the single mutated AHAS gene on 1 chromosome in the rice genome. Moreover, phytotoxicity ratings of rice event RTC1 treated with imazethapyr demonstrated consistent herbicide tolerance over multiple generations.

4. Criteria for the environmental assessment

Rice event RTC1 will not be grown in Canada and will only be imported to Canada 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 rice event RTC1 would be capable of persisting in the Canadian environment.

Potential for rice event RTC1 to become a weed of agriculture or be invasive of natural habitats

The center of origin of rice (Oryza sativa L.) is considered to be in the subtropics of Southeast Asia. Rice is not adapted to the environmental conditions encountered in most Canadian agricultural areas.

The CFIA evaluated data submitted by RiceTec Inc. on the reproductive biology and life history traits of rice event RTC1. The phenotypic and agronomic traits, such as days to 50% heading, days to maturity, stand count, plant height, grain yield, grain length, grain width, % amylose, were determined to be equivalent to the unmodified rice control variety and within the normal range of expression of the traits currently displayed by conventional 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 and growth characteristics were modified.

Rice event RTC1 is not intended for cultivation in Canada and the novel trait has no intended effects on weediness or invasiveness. The CFIA has therefore concluded that rice event RTC1 has no altered weediness or invasiveness potential in Canada.

Potential for gene flow from rice event RTC1 to sexually compatible plants whose hybrid 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 L.) belongs to a species that is not sexually compatible with domesticated rice (Oryza sativa L.). Rice event RTC1 will not be cultivated in Canada and if released, would not persist.

This information, together with the fact that the novel trait has no intended effects on rice reproductive biology, led the CFIA to conclude that gene flow form rice event RTC1 to sexually compatible species in Canada is not possible.

Potential for rice event RTC1 to become a plant pest

Rice is not a plant pest in Canada and the intended effect of the novel trait is unrelated to plant pest potential. In addition, agronomic characteristics of rice event RTC1 are similar to those described for currently commercialized rice varieties.

The CFIA has therefore determined that rice event RTC1 does not present a plant pest concern.

Potential impact of rice event RTC1 and its gene products on non-target organisms, including humans

The imidazolinone tolerance trait introduced into rice event RTC1 is unrelated to a potential impact on non-target organisms. The AHAS protein is ubiquitous in higher plants and has a history of safe consumption by humans and animals. The amino acid substitution in the AHAS protein that is present in rice event RTC1 has been introduced previously in herbicide tolerant plants that have been authorized for unconfined environmental release in Canada.

Detailed characterization of the AHAS protein expressed in rice event RTC1 led to the conclusion that this protein does not display any characteristic of a potential toxin or allergen (see Section 5: Potential impact of rice event RTC1 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed). Therefore, no negative impact resulting from exposure of organisms to the AHAS protein expressed in rice event RTC1 is expected.

Composition analyses showed that the levels of key nutrients and anti-nutrients in grain from rice event RTC1 are comparable to those in conventional rice varieties (see Section 5: Potential impact of rice event RTC1 on livestock nutrition). Therefore, it is very unlikely that the introduction of the novel trait may have caused unintended changes to the composition of rice event RTC1 that would negatively impact organisms interacting with rice event RTC1.

Rice event RTC1 will not be grown in Canada. In the event that grain from rice event RTC1 is accidentally released into the environment, it is unlikely that it would be capable of germination and growth. Therefore, exposure of non-target organisms to the AHAS protein from rice event RTC1 is expected to be minimal to non-existent.

Based on the above, the CFIA has determined that rice event RTC1 will not result in altered impacts on interacting organisms, including humans, when compared to current rice varieties.

Potential impact of rice event RTC1 on biodiversity

Rice is not adapted to the environmental conditions encountered in most Canadian agricultural areas. In addition, rice event RTC1 has no observed or expected modifications that would allow them to better survive in the Canadian environment and, as a result, are unlikely to enter or survive in managed or unmanaged ecosystems in Canada. Furthermore, there is no evidence to suggest that rice event RTC1 would cause negative impacts on interacting organisms.

The CFIA has therefore concluded that rice event RTC1 does not present any adverse impacts on biodiversity in Canada.

5. Criteria for the livestock feed assessment

The Animal Feed Program considered the nutrient and anti-nutrient profiles of rice event RTC1. It also took into consideration the safety of feed ingredients derived from rice event RTC1, including the presence of gene products, residues and metabolites in terms of animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed. Finally, the Animal Feed Program sought to determine whether feeds derived from rice event RTC1 meet the definitions and requirements of feeds as listed in Schedule IV of the Feeds Regulations.

Potential impact of rice event RTC1 on livestock nutrition

Nutritional and anti-nutrient composition

The nutritional equivalence between rice event RTC1 (sprayed with imazethapyr) and the unmodified control rice varieties (Cheniere and Roy) was determined from 4 replicated field trials with 3 trials in Argentina and 1 in Brazil during the 2015 to 2016 growing seasons. Trials were planted in a randomized complete block design. Seed was planted using standard commercial methods and rates of nitrogen fertilizer application. Imazethapyr was applied twice at a rate of 70.6 gai/ha following the standard practices for the tested hybrids. Paddy rice samples collected were analyzed for proximate, fiber, amino acids, fatty acids, minerals, vitamins, and anti-nutrients (phytic acid, trypsin and lectin) as recommended by the Organisation for Economic Co-operation and Development (OECD) consensus document for new varieties of rice (OECD, 1999).

Composition data was analyzed statistically using R-Excel version 3.2.16 and statistical differences observed between rice event RTC1 and the unmodified control rice varieties were identified (P<0.05). The biological relevance of any significant difference between rice event RTC1 and the unmodified control rice varieties was assessed within the range of the mean between rice event RTC1 and the unmodified control rice varieties.

There were no statistically significant differences between the unmodified control rice varieties and rice event RTC1 whole seeds for crude fat, ash, acid detergent fiber (ADF), total dietary fiber (TDF) and amylose. Statistically significant differences were found between seeds from rice event RTC1 and unmodified control rice varieties for crude protein, neutral detergent fiber (NDF) and carbohydrates. However the means were within the range of the mean between rice event RTC1 event and the unmodified control rice varieties.

There were no statistically significant differences between the unmodified control rice varieties and rice event RTC1 whole seeds for phosphorus, iron, magnesium, and copper. Statistically significant differences were found between seeds from rice event RTC1 and unmodified control rice varieties for calcium, potassium, manganese and zinc. However the means were within the range of the mean between rice event RTC1 and the unmodified control rice varieties.

There were no statistically significant differences between the unmodified control rice variety and rice event RTC1 whole seeds for niacin, vitamin B6, folic acid, and biotin. Statistically significant differences were found between seeds from rice event RTC1 and unmodified control rice varieties for thiamine and pantothenic acid. However the means were within the range of the mean between rice event RTC1 event and the unmodified control rice varieties.

There were no statistically significant differences between the unmodified control rice varieties and rice event RTC1 whole seeds for aspartic acid, threonine, glycine, cystine, methionine, lysine, histidine, arginine and tryptophan. Statistically significant differences were found between seeds from rice event RTC1 and unmodified control rice varieties for serine, glutamic acid, proline, alanine, valine, isoleucine, leucine, tyrosine and phenylalanine. However the means were within the range of the mean between rice event RTC1 and the unmodified control rice varieties.

Statistically significant differences were noted for stearic, oleic, behenic acid, and lignoceric between seeds of rice event RTC1 and the unmodified control rice varieties. However, the means were within the range of the mean between rice event RTC1 and the unmodified control rice varieties grown in the trial; and therefore the differences were not considered biologically relevant. No significant differences were observed between the unmodified control rice varieties and rice event RTC1 for myristic, palmitic acid, palmitoleic acid, linoleic acid, linolenic acid, arachidic acid, eicosenoic acid, eicosadienoic acid, erucic and tricosanoic acids.

No significant differences were observed between the unmodified control rice varieties and rice event RTC1 for all anti-nutrients analyzed.

Conclusion

It was concluded based on the evidence provided by RiceTec Inc. that the nutritional composition of rice event RTC1 is similar to that of the unmodified control rice varieties.

Potential impact of rice event RTC1 on animal health and human safety as it relates to the potential transfer of residues into foods of animal origin and worker/bystander exposure to the feed

Rice event RTC1 displays tolerance to imidazolinone due to a point mutation introduced in the AHAS gene resulting in a modified AHAS protein that is not susceptible to imidazolinone. The assessment of rice event RTC1 used the weight of evidence approach to evaluate the risk of the modified AHAS to livestock consuming feed ingredients from rice event RTC1, humans consuming foods of animal origin derived from those livestock, and worker/bystander exposed to the feed ingredients from this event.

Modified AHAS protein

The potential allergenicity and toxicity of the modified AHAS protein were evaluated. With respect to its potential allergenicity, the original sources of the trait, rice line P1003, is not known to produce allergens and a bioinformatics analysis of the modified AHAS protein amino acid sequence confirmed the lack of relevant similarities between this protein and known allergens. In addition, there is a history of safe use of the source organism and safe exposure to the AHAS protein. Unlike many allergens, the E. coli-produced AHAS protein is rapidly degraded in simulated gastric fluid and is not heat stable. Therefore, the weight of evidence indicates that this modified AHAS protein is unlikely to be an allergenic concern.

In terms of the potential toxicity, the modified AHAS protein lacks a mode of action to suggest that it is intrinsically toxic and a bioinformatics analysis of its protein amino acid sequence confirmed the lack of relevant similarities between this modified AHAS protein and known toxins. In addition, there is a history of safe use of the source organism and safe exposure to the AHAS protein. Furthermore, the exposure to this modified AHAS protein is expected to be low considering: 1) The AHAS protein is not expressed at a high level in conventional type rice lines and there were no exogenous promoters introduced or changes to the endogenous promoters in the rice event RTC1 to alter protein expression; 2) the E. coli-produced AHAS protein is rapidly degraded in simulated gastric fluid and is not heat stable. The weight of evidence thus indicates that this modified AHAS protein is unlikely to be toxic to livestock.

Conclusion

It was concluded, based on the evidence provided by RiceTec Inc., that the herbicide tolerance trait resulting from the modified AHAS does not confer to rice event RTC1 or any rice lines derived from it any characteristics that would raise concerns regarding its safety. Feed ingredient(s) derived from rice event RTC1 are considered to meet present ingredient definitions for rice in the Feeds Regulations and as such are approved for use as livestock feed in Canada.

6. New information requirements

If at any time RiceTec Inc. becomes aware of any new information regarding risk to the environment, livestock or human health which could result from the unconfined environmental release or livestock feed use of rice event RTC1 or lines derived from it; RiceTec Inc. is required to immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of rice event RTC1 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 rice event RTC1.

7. Regulatory decision

Rice event RTC1 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.

Based on the review of the data and information submitted by RiceTec Inc. and input from other relevant scientific sources, the Animal Feed Program has concluded that the novel AHAS protein-based herbicide tolerance trait will not confer to rice event RTC1 any characteristic that would raise concerns regarding the safety or nutritional composition of rice event RTC1. Livestock feeds derived from rice are currently listed in Schedule IV of the Feeds Regulations. Rice event RTC1 has been found to be as safe and nutritious as currently and historically grown rice varieties. Rice event RTC1 and its products are considered to meet present ingredient definitions and are therefore approved for use as livestock feed ingredients in Canada.

Taking into account this evaluation, use as livestock feed of rice event RTC1 is therefore authorized by the Animal Feed Program of the Animal Health Directorate as of June 4, 2019. Any rice lines derived from rice event RTC1 may also be used as livestock feed, provided that:

• no inter-specific crosses are performed
• the intended uses are similar
• it is known based on characterization that these plants do not display any additional novel traits and are substantially equivalent to currently commercialized rice and permitted to be used as livestock feed in Canada, in terms of their potential environmental impact and livestock feed safety
• the novel gene is expressed at a level similar to that of the authorized line

Rice event RTC1 is subject to the same phytosanitary import requirements as the unmodified rice varieties, and is required to meet the requirements of other Canadian legislation including, but not limited to, the Food and Drugs Act and the Pest Control Products Act.

Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of rice event RTC1.

Date modified:

2022-03-11