Novel food information: Cotton Event MON 88702
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Health Canada has notified Monsanto Canada Inc. that it has no objection to the food use of refined, bleached, and deodorized (RBD) cottonseed oil and cottonseed linters derived from MON 88702 cotton. The Department conducted a comprehensive assessment of this 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 notification from Monsanto Canada Inc. and the evaluation by Heath Canada and contains no confidential business information.
Monsanto Canada Inc. developed insect-protected MON 88702 cotton to protect against feeding damage caused by targeted insect pests. Recombinant DNA techniques were used in order to make the cotton insect-protected through the insertion of an expression cassette coding for a modified insecticidal crystal (Cry) protein derived from Bacillus thuringiensis ( Bt) that protects against feeding damage caused by targeted hemipteran and thysanopteran pests, including two species of tarnished plant bugs (Lygus hesperus, Lygus lineolaris), cotton fleahopper (Pseudoalomoscelis seriatus), and thrips ( Frankiniella spp.). The modified protein has been assigned the unique name Cry51Aa2.834_16 and is also referred to as mCry51Aa2. The protein differs from the wild-type Cry51Aa2 from Bt in 8 amino acid substitutions and 3 amino acid deletions, for a 96% sequence similarity.
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 MON 88702 cotton was developed; the composition and nutritional quality of RBD cottonseed oil and cottonseed linters derived from MON 88702 cotton compared to non-modified varieties; and the potential for MON 88702 cotton to be toxic or cause allergic reactions. Monsanto Canada Inc. provided data that demonstrates that RBD cottonseed oil and cottonseed linters derived from MON 88702 cotton is as safe and of the same nutritional quality as traditional cotton 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). Food use of MON 88702 cotton is considered a novel food 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 MON 88702 cotton and data that characterize the genetic modification which results in the insect-protection through the expression of an mCry51Aa2 cassette. The mCry51Aa2 protein in MON 88702 has a similar mode of action to numerous Bt Cry proteins that have been previously approved by Health Canada and the CFIA such as Cry1Ac and Cry2Ab2 in cotton. MON 88702 cotton was produced by Agrobacterium -mediated transformation of cotton tissue using the vector PV-GHIR508523 which contains two separate transfer DNA (T-DNA) cassettes.
The first T-DNA, T-DNA-I, contains the mCry51Aa2 expression cassette which is composed of a region from the 35S RNA figwort mosaic virus, a promoter and 5’ UTR leader sequence for the heat shock protein 81-2 (Hsp81-2) from Arabidopsis thaliana, the coding sequence of the mCry51Aa2 protein, and a 3’UTR sequence of the 35S RNA of cauliflower mosaic virus. The second T-DNA, T-DNA-II, contains the aadA expression cassette that is used for selection and is not present in the final line after subsequent breeding, segregation, selection and screening following transformation.
3. Characterization of the modified plant
A combination of next generation sequencing (NGS), PCR, and bioinformatics was performed in order to characterize the number of insertion sites and copies of the integrated T-DNA-I as well as the presence or absence of plasmid backbone or T-DNA II sequence. This analysis demonstrated a single copy inserted at a single locus of the cotton genome. The analysis also detected no vector backbone or T-DNA II sequences present in MON 88702 cotton.
The T-DNA-I insertion site was assessed using bioinformatics analysis to examine the potential for formation of new open reading frames (ORFs) at the insertion site in the cotton genome. A total of 10 putative polypeptides spanning the cotton genomic DNA and T-DNA-I junctions were analysed from stop codon to stop codon for all six reading frames. In the unlikely occurrence that any of the ten putative polypeptides analyzed would be found in MON 88702 cotton, there was no significant similarity shared to known allergens, toxins, or other biologically active proteins after comparing translated sequences to the allergen AD_2017 database, the toxin TOX_2017 database, and the protein PRT_2017 database using bioinformatics tools.
Next generation sequencing of five breeding generations demonstrated that the T-DNA-I insert was maintained, confirming stability. Segregation data confirmed that the insert followed the expected Mendelian inheritance patterns, which supports the genomic stability of the insert at a single locus in MON 88702 cotton.
4. Product information
One new protein would be expressed in MON 88702 cotton based on the characterization of the inserted genetic material. The mean expression levels of mCry51Aa2 protein were highest in over season leaf 1 at 1200 ug/g dw and lowest in pollen at 2.6 ug/g dw. The mCry51Aa2 protein produced in MON 88702 cotton by the insertion of T-DNA-I was characterized and compared to Bt-produced mCry51Aa2 given that a microbial expression system was used to obtain enough novel protein for the toxicological testing. This characterization included SDS-PAGE analysis, N-terminal sequencing, MALDI-TOF mass spectrometry, Western blot analysis, functional activity, and glycosylation analysis. The results demonstrated that the MON 88702 cotton produced mCry51Aa2 protein and the Bt-produced mCry51Aa2 protein are equivalent.
5. Dietary exposure
The genetic modification of MON 88702 cotton is not intended to alter the consumption of food products derived from cotton when compared to conventional non-genetically modified varieties. Human dietary exposure to cottonseed is primarily limited to refined, bleached, and deodorized (RBD) oil used in frying, salad dressings, mayonnaise, margarine, shortenings, and other food products. Cottonseed linters are short fibers removed from the cottonseed during delinting, which can be processed as a source of food grade cellulose. It is expected that RBD oil and linters from MON 88702 cotton will be used in applications similar to RBD oil and linters from conventional cotton varieties and no change in the food use of RBD oil or linters is anticipated.
Five field trials were used to collect samples for compositional analysis of MON 88702 and its near-isogenic, non-transgenic control counterpart (DP393). The field trials were conducted in the cotton growing regions of the United States in 2015. No reference lines were included in the field trials. Starting seeds were planted in a randomized complete block design with four replicates at each site. Typical commercial agriculture production practices were used for the field trials.
Acid-delinted cottonseed samples were analyzed for moisture, protein, fat, ash, total dietary fibre, neutral detergent fibre, acid detergent fibre, carbohydrate (by difference), amino acids, fatty acids, vitamin E (α-tocopherol), minerals and anti-nutrients (free and total gossypol, and malvalic acid, sterculic acid and dihyrosterculic acid,). The compositional nutrients and anti-nutrients measured were in line with the recommendations in the OECD for comparative assessment of the composition of new varieties of cotton (OECD, 2009). The analysis for each component was conducted on all samples by a single laboratory using internationally approved and validated analytical methods. The petitioner also followed consistent and appropriate sample storage and preparation procedures.
Of the 65 nutrient and anti-nutrient components of acid-delinted cottonseed measured, eight had more than 50% of the observations below the assay limit of quantitation (LOQ), and were excluded from statistical analysis. Moisture values were used for conversion to dry weight but were not statistically analyzed. Therefore, the remaining 56 components in cottonseed samples were statistically assessed using a mixed model of analysis of variance method.
Where a statistically significant difference (p-value <0.05) was identified in the combined site analysis, further context for interpreting the possible biological significance of the difference was gathered through comparisons with the range of values for each analyte reported in the published literature and/or available from the ILSI Crop Composition Database (ILSI, 2014).
There were no statistical differences for 47 of the 56 components analyzed including anti-nutrients. The nine components that were statistically different in MON88702 vs control were protein, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, arachidic acid, behenic acid, and calcium. The differences in the cottonseed protein and fatty acid levels were small (<10%) and would not impact nutritional quality of the cottonseed. The differences in individual fatty acids mean % of total fatty acids in MON88702 vs. control were all less than ±2 % and these mean differences between MON 88702 and the conventional control were less than the conventional control range values, so these differences would not be considered to be nutritionally relevant. The total fat content of MON88702 cottonseed was not different from control. The higher mean level of calcium in MON88702 vs. control (difference of 0.011% dw) was also less than the conventional control range values and thus within the natural range of variability. This could potentially be due to variation in soil mineral content among field sites. Levels of all analytes were within the range reported in the literature and/or ILSI crop composition database, except for lauric acid (difference between test and control means was only 0.002%), for which no values were available. These results indicate that there are no nutritional safety concerns with MON 88702.
Based on the available information, MON 88702 cotton is similar in composition to its conventional counterpart.
The petitioner submitted studies to support the safety of the mCry51Aa2 protein. Studies were conducted in accordance with Good Laboratory Practices (GLP) and considered acceptable for the purpose of the safety assessment.
A surrogate protein produced in a bacterial expression system was used to conduct the safety studies. The petitioner provided information to confirm the equivalence of the microbially-derived protein with the plant protein. This included assessment of molecular weight, immunoreactivity, lack of glycosylation, amino acid sequence and functional activity.
Microbially-derived mCry51Aa2 protein did not produce any adverse effects in an acute oral toxicity study in CD-1 mice (10 animals/sex/group). The results demonstrated that ingestion of mCry51Aa2 protein would not result in overt toxic effects at doses up to 5000 mg/kg body weight, the highest dose tested and the limit dose of the test.
The petitioner provided the results of an in silico analysis comparing the mCry51Aa2 amino acid sequence to proteins identified as toxins in the GenBank (217) database using the FASTA alignment tool. There was no relevant similarity to known toxins that would indicate the mCry51Aa2 protein is toxic in mammals.
Results of an in vitro simulated sequential gastric and intestinal fluid assays demonstrated that microbially-derived mCry51Aa2 was rapidly digested. This suggests that any ingested mCry51Aa2 protein is unlikely to persist in the mammalian gastrointestinal tract and therefore unlikely to exert a systemic toxic effect.
The results of a heat stability assay showed that microbially-derived mCry51Aa2 protein was inactivated at temperatures commonly involved in cotton oil processing (> 55 ºC for 15 minutes). This finding suggests that consumers would not be exposed to an active protein.
Dietary exposure to cottonseed is primarily limited to refined, bleached and deodorized (RBD) oil and cottonseed linters used as a source of food grade cellulose. Both products are highly processed and expected to contain very low amounts of protein. Results from field expression studies provided by the petitioner showed that the mCry51Aa2 protein represents a minimal amount (0.05%) of total seed protein. Therefore, the dietary exposure to the mCry51Aa2 protein from MON 88702 was considered negligible.
Given that most allergens are proteins and human dietary exposure is limited to cottonseed oil and cotton linters that contain negligible protein, these products would not be expected to pose an allergic risk.
The petitioner provided an in silico sequence analysis of the novel mCry51Aa2 protein against 1,970 known allergens in an allergen, gliadin and glutenin sequence database (COMprehensive Protein Allergen REsource 2017 database from the Health and Environmental Sciences Institute) using the FASTA alignment tool. The search included an eight amino acid sliding window as well as sequences with a linear identity of > 35% in an 80 amino acid overlap to compare the mCry51Aa2 protein to known allergens. No matches were identified for the mCry51Aa2 protein.
The petitioner provided experimental data that demonstrated that microbially-derived mCry51Aa2 protein is readily digestible in simulated gastric fluid based on SDS-PAGE and Western blot analysis. This suggests that any ingested mCry51Aa2 protein is not readily available to provoke an allergic reaction.
Based on the above, RBD cottonseed oil and cottonseed linters derived from MON 88702 cotton are not considered to pose a greater toxicological or allergenic risk than conventional cotton varieties.
Health Canada’s review of the information presented in support of the food use of RBD cottonseed oil and cottonseed linters from MON 88702 cotton does not raise concerns related to food safety. Health Canada is of the opinion that food derived from MON 88702 cotton is as safe and nutritious as food from current commercial cotton varieties.
Health Canada's opinion deals only with the food use of MON 88702 cotton.
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
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
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