Novel Food Information - Cotton Producing Demethylase (DMO) and Phosphinothricin N-acetyltransferase (PAT) - MON 88701

Health Canada has notified Monsanto Canada, Inc. that it has no objection to the food use of herbicide tolerant cotton event MON 88701. The Department conducted a comprehensive assessment of this cotton event 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.

Background

The following provides a summary of the notification from Monsanto Canada, Inc. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

MON 88701 cotton produces two herbicide tolerance genes: a modified version of dmo which expresses demethylase (DMO), an enzyme which confers resistance to dicamba (3,6-dichloro-2-methoxybenzoic acid); and a bar gene encoding for phosphinothricin N-acetyltransferase (PAT) which confers resistance to glufosinate (2-amino-4-(hydroxymethyl- phosphinyl) butanoic acid). The herbicide tolerance was achieved through transformation of a conventional cotton variety.

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 (e.g. Codex Alimentarius). The assessment considered: how MON 88701cotton was developed; how the composition and nutritional quality of MON 88701cotton compared to non-modified varieties; and what the potential is for MON 88701 cotton to be toxic or cause allergic reactions. Monsanto has provided data that demonstrates MON 88701cotton 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 Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from MON 88701cotton are considered novel foods 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

MON 88701 was genetically modified by Agrobacterium tumefaciens-mediated DNA transfer of a transformation plasmid. The T-DNA (or insert) region of the plasmid contains the plant-optimized coding sequences for both the dmo and bar genes. Seeds from Coker 130 variety cotton, the recipient host, were germinated in the dark. The hypocotyl tissue was excised and co-cultured with Agrobacterium bearing the plasmid vector to allow callus formation. The tissue was stimulated with plant growth regulators to induce regeneration of plants. Grown plants were self-pollinated and the original transformed plants and their progeny were screened for expression of the desired traits, resistance to dicamba and glufosinate. Molecular screening by PCR and Southern blot was performed to test for presence of the dmo and bar expression cassette DNA, as well as absence of the plasmid backbone.

3. Characterization of the Modified Plant

Southern blot analyses were performed in order to confirm and characterize the genomic insertion of the dmo and bar expression cassettes in MON 88701. The integrity of the dmo and bar genes and their respective regulatory elements was shown, and the absence of plasmid backbone DNA in MON 88701 was confirmed. The stability and inheritance of the inserted cassettes was also evaluated in several generations of MON 88701 by Southern blot analysis. The results demonstrated the apparent molecular stability of the inserted cassette, i.e., the inserted DNA was intact and at a single site following a traditional breeding program. As well, the genetic segregation data showed that the DMO and PAT traits were inherited and expressed in the expected Mendelian manner. The insert region was also sequenced to demonstrate that no changes had occurred with the insertion at the nucleotide level. Sequencing of the genomic regions flanking the insert site showed that a 123 base pair deletion had occurred within the cotton genome as a result of the insertion of T-DNA in MON 88701. Deletions such as this are presumed to be the result of double-stranded break repair that can occur when using Agrobacterium-mediated DNA transformation. This unintended deletion is not expected to have any bearing on the safety of event MON 88701.

Bioinformatics analyses were performed on the DNA sequences of the insert and the adjacent genomic regions in order to determine whether potential novel open reading frames (ORFs) were inadvertently created which may show similarity to allergens, toxins, or other proteins with biological activity that could impact overall product safety. The insert region and genomic regions flanking the insertion site were sequenced and translated in all six reading frames. Predicted polypeptides of at least eight amino acids in length were searched against databases of toxins, allergens, and all proteins, and no similarities were found.

4. Product Information

MON 88701 cotton differs from traditional cotton by the addition of the coding sequences for dmo derived from Stenotrophomonas maltophilia and bar derived from Streptomyces hygroscopicus. The donor organisms are both soil bacteria and are not known to be a human pathogen. The dmo gene in MON 88701 is identical to its counterpart in S. maltophilia except that codons were modified to optimize gene expression in plants without altering the amino acid sequence, and an additional codon for leucine was inserted after the start methionine of dmo. Upstream of dmo was appended the coding sequence for a 76 residue chloroplast transit peptide from the Arabidopsis thaliana shkG gene which serves to target the DMO protein to the chloroplast. When expressed in cotton, all but the last nine residues of this peptide are removed from DMO by post-translational processing. The precursor MON 88701 DMO protein is 416 amino acid residues long, and upon maturation it is truncated to 349 residues. The codons for the bar gene were likewise optimized for plant expression. The expressed PAT enzyme is 183 amino acids in length and of identical sequence to the corresponding enzyme from the donor organism.

The demethylase enzyme DMO catalyzes the degradation of the dicamba herbicides into an inactive compound, 3,6-dichlorosalicylic acid (DCSA). Phosphinothricin N-acetyltransferase, or PAT, catalyzes the inactivation of phosphinothricin (glufosinate) by acetylation of the primary amino group.

The expression levels of DMO and PAT in MON 88701 plant tissues were determined using a validated double sandwich enzyme-linked immunosorbent assays (ELISAs). All tissues of the transgenic plants tested positive for DMO and PAT in these assay. The portion of the plant that enters the food supply is the seed, which is subject to processing. Seed harvested from MON 88701 plants contained on average 21 μg DMO/g dry weight tissue (range: 8.9 to 33 μg DMO/g) and 6.6 μg PAT/g dry weight tissue (5.2 to 9.6 μg PAT/g).

5. Dietary Exposure

The genetic modification of MON 88701cotton is not intended to alter cotton consumption patterns. Therefore, the use of herbicide tolerant MON 88701cotton and products derived from it will be similar to traditional cotton varieties. The introduction of MON 88701cotton will only replace a fraction of current cotton varieties and is not anticipated to result in a change in the dietary intake of cotton-derived products.

6. Nutrition

Levels of key nutrients and anti-nutrients were determined in cottonseed samples collected from MON 88701 and control cotton plants grown in a field trial. Cottonseeds were harvested, ginned, acid-delinted, and ground prior to analysis. Nutrients and anti-nutrients were selected according to the OECD's Consensus document on composition considerations for new varieties of cotton. Component levels between MON 88701 and the control line were compared at both individual site and combined-site levels using a mixed model analysis of variance.

Of the 65 components analyzed, 13 had more than 50% of the observations below the assay lower limit of quantification and were thus excluded from statistical analysis. In the combined-site analysis, no statistically significant differences between MON 88701 and the conventional control were found for 30 components (28 nutrients and 2 anti-nutrients).  The combined-site analysis showed significant differences between MON 88701 and the control for 4 proximates (ash, moisture, and total fat higher in MON 88701; carbohydrates lower in MON 88701), fibre (ADF, NDF, and TDF lower in MON 88701), three amino acids (methionine higher in MON 88701; arginine and proline lower in MON 88701), two fatty acids (14:0 myristic acid and 18:2 linoleic acid lower in MON 88701), five minerals (calcium, magnesium, manganese, potassium, and zinc higher in MON 88701), vitamin E (higher in MON 88701), and three anti-nutrients (dihydrosterculic acid, free gossypol, and total gossypol higher in MON 88701; discussed below).

All MON 88701 analytes that were significantly different from the control had a relative mean difference <10% when compared to the control; the only exception being calcium with a relative mean difference >10% (approximately 14% higher in MON 88701).With the exception of methionine (MON 88701: 0.40 % dw, Control: 0.38 % dw, 99% tolerance interval: 0.32-0.38), all combined-site mean values of MON 88701 were within the 99% tolerance interval established from the conventional commercial reference varieties grown in the same trial. All combined-site mean values and ranges of MON 88701 for all nutrient components, including those that were significantly different, were within the context of the natural variability of commercial cotton composition as published in the scientific literature and/or available in the ILSI Crop Composition Database.

It was found that the quantity and quality of the information provided by the petitioner was satisfactory. Overall, the data showed that the nutritional composition of cottonseed from MON 88701 is comparable to cottonseed from a near isogenic, non-modified parent and did not raise any nutritional or safety concerns.

7. Chemistry/Toxicology

Based on the compositional comparative analysis, mean values of dihydrosterculic acid, free gossypol, and total gossypol in MON 88701 were 9.59 %, 6.23 %, and 6.75 % higher, respectively, relative to the conventional control. These differences were determined by the petitioner to be within the 99 % tolerance interval established by both the conventional commercial reference varieties grown concurrently and the natural variability of commercial cotton composition as reported in the scientific literature. Given that the differences in gossypol levels between MON 88701 cottonseed and conventional cotton varieties are minor, it is expected that the levels of gossypol in processed oil and flour from these lines would be similar, assuming they are subjected to the same processing conditions.

Given the mode of action for the herbicide tolerance, modification of this plant through insertion of herbicide tolerance genes is not expected to alter a plant's susceptibility to fungi that produce mycotoxins, nor does it increase the uptake or storage of chemical contaminants. As is generally the case for plant events expressing an herbicide tolerance trait, mineral data provided was related to nutrition rather than chemical contamination, and data on the presence of mycotoxins was not provided. Based on the understanding of the DMO and PAT herbicide tolerance functions, no concerns were identified regarding the food use of MON 88701cotton from a chemical contaminants perspective.

A multistep approach was used to demonstrate the safety of DMO and PAT expressed in MON 88701. This included: documentation of the history of use of these proteins in genetically modified plants; characterization of the proteins' physicochemical and functional properties; quantification of protein expression levels in plant tissues; comparison of the protein coding sequences within the insert region to known allergens and toxins; evaluation of the proteins' digestibility in simulated gastrointestinal and intestinal fluids (SGF and SIF); evaluation of protein stability to heat treatment typical of manufacturing of food ingredients and products; and an assessment of potential allergenicity of the expressed proteins.

It was not possible to isolate purified DMO or PAT from MON 88701 in sufficient quantities to perform the required tests of physicochemical properties and toxicological testing. Versions of the proteins were therefore expressed in E. coli. DMO and PAT were each purified from both the plant and bacterial sources, and a series of tests were performed in order to demonstrate equivalence in the two expression systems.

The identities of the DMO and PAT proteins expressed in both plants and bacteria were confirmed using mass spectrometry (MS) fingerprinting, N-terminal and tryptic peptide sequencing (PAT only), electrophoresis, immunological staining. Glycosylation analysis showed that neither DMO nor PAT was post-translationally modified with sugars. Enzyme activity assays were carried out as well to determine the reaction rate constants of the expressed proteins. It was shown that, as expected, the N-terminal chloroplast transit peptide appended to DMO is removed by proteolytic processing as part of the maturation process in plants. Based on the protein characterization assays which together showed equivalency, it was considered justified to extrapolate the results of studies on protein stability and toxicity using the E. coli-expressed MON 88701 DMO and PAT to the properties of the plant-expressed version of these proteins.

Separate acute oral toxicity studies with DMO and PAT protein were provided in the petition. In the DMO acute study, E. coli-derived DMO protein was administered by gavage to CD-1 mice (n=10/sex) at a dose of 283 mg/kg bw in a single dose. A protein control group (n=10/sex) was similarly administered a single gavage dose of bovine serum albumin at dose equivalent to 280 mg/kg bw. Animals were observed two or more times daily for clinical signs of toxicity, mortality or moribundity while food consumption was measured daily. Body weights were recorded at time of randomization, prior to fasting and dosing and on days 3, 7, 10 and 14. At the end of the study on day 14, animals were euthanized by CO2 and exsanguination prior to necropsy. No treatment-related adverse effects, pathology findings or mortality were observed in DMO-treated animals. Body weight, body weight gain and food consumption were not different from the protein control group. Based on a lack of adverse effects or mortality, the NOEL was considered to be 283 mg/kg bw.

E. coli-derived PAT protein was administered by gavage a group of CD-1 mice (n=10/sex) at a dose level of 1086 mg/kg bw as a single dose. A protein control group (n=10/sex) received a comparable dose of bovine serum albumin, equivalent to 1012 mg/kg bw. Animals were observed two or more times daily for clinical signs of toxicity, mortality or moribundity while food consumption was measured daily. Body weights were recorded at randomization, prior to fasting and dosing and on days 3, 7, 10 and 14. At the end of the observation period on day 14, all animals were subject to necropsy. No treatment-related adverse effects, pathology findings or mortality were observed in PAT-treated animals. Body weight, body weight gain and food consumption were not different from the protein control group. The NOEL for this study was considered to be 1086 mg/kg bw, the highest dose tested.

The petitioner also provided evidence supporting that DMO and PAT proteins are readily digested in the stomach and intestinal tract using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) studies. In the SGF study, both proteins derived from E. coli were completely digested by pepsin by 0.5 minutes based on SDS-PAGE and Western blot analyses with the appropriate controls. Additionally, digestion tests with SIF indicated that both DMO and PAT proteins can be digested within 0.5 minutes, suggesting that any ingested DMO or PAT proteins would be degraded once past the stomach, making it of low concern for allergenicity.

The thermal stability of the DMO and PAT was evaluated by heating to 25, 37, 55, 75 or 95 °C for 15 or 30 minutes followed by a functionality assay and SDS-PAGE analysis to determine protein integrity. DMO protein functionality was decreased below the limit of detection after heat treatment at 55 °C or higher for 15 or 30 minutes. SDS-PAGE showed no differences in band intensity at any time point or temperature. PAT protein functionality was also reduced to 9 % of control levels at 95 °C for 15 or 30 minutes although band intensity in the SDS-PAGE analysis was not reduced at any temperatures. Therefore, while functionality of the proteins was reduced after high heat, protein integrity was maintained at all temperatures tested. It can, however, be considered of low significance in light of the negligible amount of estimated protein in highly refined cottonseed oil (see below).

The amino acid sequences of DMO and PAT proteins were evaluated using FASTA bioinformatic alignment searches performed with 10,570 sequences in the TOX_2011 database; a subset of sequences derived from the PRT_2011 database using keyword specific exclusion criteria to retain relevant proteins and toxins. Based on the E-score, no structural similarities exist between DMO or PAT proteins and known toxins or biologically active harmful proteins.

Human dietary exposure to cottonseed is primarily limited to refined, bleached and deodorized cottonseed oil used in frying, salad dressings, margarines, shortenings and other food products. Due the high degree of refinement of the oil, a negligible amount of protein is present. The measured percentages of DMO and PAT protein in MON 88701cottonseed were 0.008 % and 0.002 % of total protein, respectively, while total protein in oil derived from cottonseed was undetectable. The negligible amount of DMO and PAT protein in oil produced from MON 88701 cottonseed would be heat denatured by the refining process, and this was demonstrated using enzyme function assays.

Cottonseed linters consisting of >99% cellulose are by-products of cotton processing and have food uses including fibre supplements, casings for processed meats, pharmaceutical bindings and viscosity enhancers. However, like the refined oils, linters contain negligible protein and are therefore not expected to add to the dietary exposure of DMO and PAT proteins and a margin of exposure was not calculated. As the exposure to dietary DMO and PAT protein from all sources of food derived from MON 88701 is negligible, the possibility of a risk to health was considered remote.

8. Allergenicity

The potential allergenicity of MON 88701 was determined through a comparison between the amino acid sequences of DMO and PAT proteins to known allergens. Guidelines proposed by Codex Alimentarius (2009) state that cross-reactivity may be present if the introduced protein has ≥35 % amino acid identity with an allergen over a segment of ≥80 amino acids. The FASTA sequence alignment tool was used to compare the amino acid sequences of MON 88701 proteins, known allergens, gliadins and glutenins, which allows for an approximation of shared secondary and tertiary structure. Amino acids sequences from DMO and PAT proteins were compared to the Allergen, Gliadin and Glutenin Sequence Database (AD_2011) from the Food Allergy Research and Resource Program Database (FARRP_2011). Results from 1491 reference sequences in the AD_2011 indicate no sequence homology between MON 88701 DMO and PAT proteins and known allergens, and no alignments exceeding the 35% amino acid threshold. An eight-amino acid sliding window search was also used to identify short amino acid sequences to known allergens, gliadins and glutenins. When compared to the AD_2011, neither protein shared any 8 amino acid sequences.

Conclusion

Health Canada's review of the information presented in support of the food use of MON 88701 cotton does not raise concerns related to food safety. Health Canada is of the opinion that food derived from MON 88701 is as safe and nutritious as food from current commercial cotton varieties.

Health Canada's opinion deals only with the food use of MON 88701 cotton. 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 herbicide tolerant cotton event MON 88701. 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 herbicide tolerant cotton event MON 88701.

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.

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
novelfoods-alimentsnouveaux@hc-sc.gc.ca