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Safety Assessment of NewLeaf Plus Potatoes Protected Against Colorado Potato Beetle and Infection by Potato Leafroll Virus

Executive Summary The Monsanto Company has developed the NewLeaf Plus potato variety that is resistant to infection by Potato Leafroll Virus (PLRV) and to feeding by the Colorado potato beetle, Leptinotarsa decemlineata (CPB). This disease and insect account for much of the need to apply insecticides to potatoes in major production areas of the United States. The Russet Burbank potato cultivar, which is the dominant potato variety in the Pacific Northwest, is particularly sensitive to PLRV and up to 100% of the crop can be infected if insecticides are not properly applied to control the aphid that vectors the virus from plant to plant. Unlike conventional Russet Burbank, tubers harvested from NewLeaf Plus do not develop the defect, net necrosis (internal browning) that is caused by PLRV infection and which makes the tubers less desirable for processing into frozen products, such as frozen french fries. To develop the NewLeaf Plus potato variety, a Russet Burbank potato line (RB1) was modified using the Agrobacterium tumefaciens transformation system. A plasmid vector was employed that contained genes with resistance to the two important potato pests: 1) Colorado potato beetle (the gene cry3A that produces a protein toxic only to target insects, isolated from the bacteria Bacillus thuringensis subsp. tenebrionis, Btt); and 2) Potato Leafroll Virus (the gene PLRVrep, isolated from a naturally occurring strain of PLRV, encodes replicase, a protein that enables virus replication in plant cells). There is also a marker gene for plant selection (nptII isolated from soil bacteria) inserted to aid for selection of modified plant cells. The gene products produced by the cry3A and PLRVrep genes provide for effective control of damage caused by CPB and PLRV respectfully by inhibiting CPB feeding on potato foliage and by inhibiting the replication of PLRV in potato tissue. Resulting potato plants were evaluated during four years of greenhouse and field tests designed to study agronomic performance and fit in conventional potato production systems. They were also evaluated for disease and pest resistance, tuber yield and quality, and conformity to variety type. The safety assessment of NewLeaf Plus potato products focused on 1) the safety of the gene products expressed, 2) the composition of tubers derived from NewLeaf Plus potato plants, and 3) assessing the environmental impact from release of these plants into the environment via commercial production as compared to conventional varieties. The presence of PLRVrep, cry3A and nptII genes or expressed proteins was determined by molecular or biochemical methods. Intact copies of the genes were demonstrated to be present and to express anticipated products (detectable levels of mRNA or proteins).

NewLeaf is a registered trademark of Monsanto Technology LLC.

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The mechanism of action for the Cry3A protein produced by B. thuringiensis subsp. tenebrionsis (Btt) and by NewLeaf potato plants has been well defined. Ingestion of the Cry3A protein produced by NewLeaf potato plants stops insect feeding eventually leading to death by starvation. The mechanism of feeding inhibition relies on highly specific binding of the Cry3A protein to cells lining the insect gut. The ligand-receptor based binding causes a disruption in ion flow leading to pore formation in the cells thus destroying the integrity of the gut cells. The receptors for the Cry3A protein are not present on mammalian cells nor in insects not closely related to CPB. Hence the Cry3A protein is effective for control of a limited number of coleopteran insects and only those insects that might feed on potato tissue produced by NewLeaf potato plants. Expressed by the plant, the PLRVrep gene prevents invading PLRV from establishing and causing disease. The exact mechanism for resistance is not entirely understood. The protein products of these genes were determined not to be toxic nor allergenic through a history of safe use and a series of scientific studies. PLRV replicates in the vascular tissues of the potato tuber; therefore, humans and animals have routinely consumed PLRV virons and PLRV replicase protein, without deleterious health effects. Spores of Bt, which contain the insect toxic protein, are widely used in commercial agriculture globally and are demonstrated to be safe for humans and the environment. NPTII protein is produced by microorganisms normally present in mammalian intestines. Composition and nutritional properties of NewLeaf Plus potato tubers are equivalent to the parental variety and within the scientific literature-established range for white potatoes. Levels of naturally occurring potato toxins (glycoalkaloids) found in NewLeaf Plus tubers are typical of those found in comparable potatoes. The environmental impact assessment from the release of NewLeaf Plus potatoes took into consideration whether NewLeaf Plus potatoes showed any altered plant pest characteristics beyond those associated with comparable conventional parental varieties. There is no meaningful potential for pollen mediated gene flow to weedy relatives or conventional potato plants because there are no wild relatives of potatoes native to production areas of North America to which these potatoes could cross-pollinate and produce viable seed. Also, NewLeaf Plus plants exhibit the male-sterile characteristic of the parental variety Russet Burbank reducing the potential for pollen flow to essentially zero. Field trials of these potatoes demonstrated that volunteer potatoes, should they emerge from harvested fields, are easily controlled using standard agricultural methods. Yield and pest resistance (other than the introduced trait) are within the anticipated range for the commercial potato varieties. The protein produced by the introduced cry3A gene is rapidly degraded in the environment following harvest. The protein produced by the PLRVrep gene is vanishingly small and at a level below the limit of detection. An insect resistance management program was implemented in support of the commercial introduction of potatoes expressing Bt genes because CPB has a history of developing resistance to chemical pesticides. In the three years of commercial use of NewLeaf Plus potatoes or the five years of commercial use of NewLeaf potatoes there were no confirmed reports of CPB resistance development.

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Introduction The Monsanto Company has developed NewLeaf Plus potato that is very resistant to infection by Potato Leafroll Virus (PLRV) and to feeding by the Colorado potato beetle Leptinotarsa decemlineata (CPB) (Lawson et al.2001; Thomas et al. 2000). To develop these potatoes, select clones of the Russet Burbank potato variety were supplemented with the cry3A gene isolated from a naturally occurring soil bacterium and the PLRVrep gene isolated from a naturally occurring strain of the potato leaf roll virus. The extensive safety data package generated on the gene expressed proteins, agronomic performance of the plants, and composition of the tubers from the NewLeaf Plus potato plants supports the finding that these potatoes are no different from conventional potatoes of the same varieties except for the introduced traits, both of which were shown to raise no safety concerns (Betz et al. 2000; Kaniewski and Thomas 1999; Lavrik et al. 1995). NewLeaf Plus plants are phenotypically indistinguishable from the parental variety Russet Burbank. The potato tubers from the NewLeaf Plus potato plants are as wholesome for food and feed as conventional potato tubers in commerce. In conventional potatoes, infection by PLRV in the Russet Burbank variety is associated with tuber net necrosis, a visible browning or “netting” of tuber tissue that lowers tuber quality. Although acceptable for consumption, PLRV infected tubers are rejected by food processing companies because visible cosmetic defects in frozen potato products arise from net necrosis. Since most Russet Burbank potatoes are grown for the process market, PLRV infected fields experience marketable yield losses as high as 50-80% (Banttari et al., 1993). Even at 6% tuber damage, losses can reach $2500/ha (Reibe and Zalewski , in press). Standard conventional practices for control of PLRV include the planting of seed tubers certified to be free of PLRV infection and season-long applications of insecticides to control the aphid that vectors this virus. However, even with use of certified seed and diligent efforts to control the in-season spread of the virus in commercial production, up to 10 - 15% of the potatoes produced are infected with PLRV (Thomas 1983; Thomas et al. 1997). NewLeaf Plus potatoes contribute value to potato producers through reduction in the amount of pesticides needed to prevent damage from PLRV and CPB (Reibe and Zalewski, in press). In 1998, the authors found that NewLeaf Plus potato use reduced insecticide and miticide applications by 80% over that for conventional farmed Russet Burbank in the Columbia Basin growing region. This represents a potential elimination of a significant portion of the 500,000kg of insecticide active ingredient applied to the 35,000ha of Russet Burbank grown in the Columbia Basin alone. Individual NewLeaf Plus growers realized an average net savings of approximately $331/ha because of the reduction in pesticide applications and the higher quality of the harvested tuber due to the reduction in net necrosis. The reduction in tuber net necrosis in NewLeaf Plus compared to Russet Burbank was worth an average of $302/ha alone in sales returns to the grower. Carpenter and Gianessi (2001) estimated average cost savings of $85-134/acre with this technology.

The Potato and Selected Variety The potato (Solanum tuberosum) is native to the western hemisphere and occurs in abundance from the tropical highlands of Mexico, southward throughout western South

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America (OECD 1997). Around 1570, South American cultivated potatoes were introduced into Europe. In the 18th century, European potato varieties came to North America with farmers immigrating in search of better farmland. Potatoes are produced in all fifty states with the U.S. ranking fourth in world production (www.aphis.usda.gov/biotech/potato.html). The major commercial production areas are located in the northernmost states of the continental United States, including Maine, New York, Michigan, Wisconsin, North Dakota, Idaho, Oregon and Washington. Per capita consumption of potatoes in the United States is approximately 130 pounds per year or more than one 150 g potato each per day (USDA 1984). In 2000, 82% of the crop was used for human consumption (either processed or as table stock), 6% was planted as seed, and 2% was used for animal feed. Shrinkage, loss, and home use represented the remaining 10% (USDA 2001a). These use ratios have not changed significantly since before 1992 (USDA 2001b). Potatoes are vegetatively propagated, i.e. cloned. Potato certified “seeds” are actually tubers from potato crops grown specifically for propagation purposes. The tubers are genetically identical to the plant that produced them. Therefore, there is no genetic mixing or crossing within crops produced via cloning. Each potato variety was selected for desirable characteristics that distinguish the variety. Varieties are maintained through a sophisticated clonal propagation system. The following information describes the Russet Burbank variety: • Characteristics: Russet Burbank is a male sterile, tetraploid potato variety

(www.aphis.usda.gov/biotech/potato.html; http://www.potatoes.com). It is a high yielding, high specific gravity potato requiring a relatively long growing season. It is highly susceptible to Potato Leaf Roll Virus (which causes the tuber defect known as “net necrosis”).

• Parentage: Parentage goes back to the variety Burbank. Luther Burbank was the breeder of Burbank variety that was released in 1874. Lon D. Sweet selected the russet mutation.

• Description: Russet Burbank tubers are long with numerous well distributed shallow

eyes. The tubers have russeted and heavily netted skin with white flesh. Russet Burbank is classified as a table and processing variety. Principal markets include fresh tablestock and processing, particularly in the manufacture of frozen fries where it is the standard for french fry quality. Russet Burbank is the dominant potato cultivar produced in the U.S.

This variety is highly susceptible to potato leafroll virus and to damage due to feeding by the Colorado potato beetle. Aphid vectors of PLRV and the CPB are two of four major insect pests that cause US potato producers to treat their crops with insecticide (Krieg et al., 1983; Casagrande, 1987). In 1999, 93% of the 1.1 million potato acres were treated with a total of 2.6 million pounds of insecticide (USDA, 2000). To date, no traditionally bred cultivars have been produced which are resistant to the CPB and there is only limited resistance to PLRV in the popular commercial potato cultivars. On Russet Burbank potatoes, which are grown on up to 90% of the potato acres in the West, the aphid vector of PLRV is the primary target of applied insecticides (Radcliffe et al. 1991). Because infection of Russet Burbank potatoes by this virus can result in severely degraded tuber quality due to net necrosis,

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growers commonly apply insecticides from plant emergence in the spring to plant senescence in the fall following extension service recommendations of applications every 7 to 10 days for foliar insecticides (Reibe and Zalewski, in press). For traditional potato varieties, approximately one-third of the pounds of chemical insecticides annually applied to potatoes are targeted for CPB control (USDA, 1997). If poorly managed in areas of severe infestation, the CPB is capable of completely defoliating potato plants, resulting in yield reductions of as much as 85% (Hare, 1980; Ferro et al., 1983; Shields and Wyman, 1984; Reed et al., 2001). Monsanto found that a replicase gene isolated from a strain of PLRV and a specific protein produced by strain of Bacillus thuriengensis subsp. tenebrionis (Btt) provided effective control of PLRV and CPB respectively when expressed in potato plant cells. This discovery led to the development of commercial NewLeaf and NewLeaf Plus potato varieties resistant to feeding by CPB (Betz et al. 2000; Perlak, et al., 1993). Field research and commercial production of NewLeaf Plus confirmed excellent control of PLRV infection and of damage caused by CPB. In the field, these potato varieties performed as expected in regards to plant growth and yield (Thomas et al., 2000). Further, scientists concluded that the reduction of pesticide applications to NewLeaf and NewLeaf Plus plants increased populations of beneficial insects (Betz et al., 2000; Reed et al., 2001). Modification Process for potatoes Russet Burbank potato lines were transformed by inserting the desired genes using the Agrobacterium tumefaciens based bacterial transformation system. In this process, cells from one Russet Burbank parental line were supplemented with the cry3A and PLRVrep genes. These genes, in a plasmid vector (Figure 1), were carried into each plant cell by the bacterial system as described by Newell et al., 1991. The plasmid vector carried an expression cassette with genes that encode for resistance to the pests Colorado potato beetle (cry3A) and Potato Leafroll Virus (PLRVrep) and marker genes for plant selection (nptII) along with appropriate gene expression promoters (Lawson et al., 2001). The Agrobacterium used in the transformation process is unable to cause crown gall disease and is no longer considered a plant pest threat. The Agrobacterium containing the plasmid vector was added to potato stem sections in tissue culture dishes. Expression cassettes, carrying the cry3A, PLRVrep and nptII genes plus promoters, were inserted via the Agrobacterium plasmid into the genome of individual potato cells. The presence of the expressed nptII gene marked those cells that were successfully transformed and allowed the selection of these cells on kanamycin containing media. Subsequently, the selected potato cells were treated to stimulate regeneration of transgenic shoots (plantlets). Ultimately plantlets were grown in soil and assayed for CPB and PLRV resistance. A number of individual plantlet lines of the Russet Burbank variety were cloned and taken through laboratory, greenhouse, and ultimately, field screens for selection of lines that exhibited the desired characteristics (Thomas and Kaniewski, 1998).

Cry3A gene and its encoded Cry3A protein The cry3A gene was isolated from the DNA of a strain of Bacillus thuriengensis subsp. tenebrionis (Btt), a common soil bacteria used for insect-control products (McPherson et al., 1988). More than thirty years of use and testing of B. thuriengensis based products have shown that the proteins produce toxic effects only in the gut of chewing insects and are not activated in human digestive tracts.

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The cry3A gene encodes for a protein, normally produced by the bacterium during sporulation, that is toxic only to targeted leaf feeding beetles, including CPB. Upon ingestion of Cry3A protein by susceptible species, feeding is inhibited through disruption of the gut epithelium. This modified gene encodes the identical amino acid sequence of band-3 protein produced by the Btt bacterium. The protein produced by the gene is identical to that found in nature and in commercial spray formulations of Btt registered with the United States Environmental Protection Agency (EPA 1991). The Cry3A protein produced by NewLeaf Plus potatoes was granted an exemption from the requirement of a USEPA FIFRA tolerance based on a review of available information and a finding of little potential for human dietary toxicity (EPA 1995). PLRVrep gene and replicase protein PLRV replicase protein is encoded by a two part gene from the potato leafroll virus (PLRVrep). NewLeaf Plus potatoes contain the nature-identical PLRVrep gene. The product of the gene is termed replicase because it is essential for replication of the PLRV RNA genome (Mayo and Ziegler-Graf, 1996; Van der Wilk et al., 1997 ). Expressed by the plant, the PLRVrep gene prevents invading PLRV from establishing and causing disease (Lawson et al., 2001; Thomas et al., 2000). The exact mechanism for resistance is not entirely understood. An exemption from the requirement of a US EPA FIFRA tolerance for the PLRVrep gene and gene product was granted by the Agency based on the natural occurrence of the replicase gene and protein in the food supply (EPA 1997). Virus-infected plants currently are and have always been a part of both the human and animal food supply. There have been no findings that indicate that plant viruses are toxic to humans and other vertebrates. Although no PLRV replicase protein was detected in NewLeaf Plus potato plants, it was expected that much lower levels of the protein would be produced in NewLeaf Plus potato plants than in Russet Burbank naturally infected with the virus. Messenger RNA (mRNA) encoding for replicase proteins was produced in NewLeaf Plus at levels well below that occurring in infected potato plants. The published literature established that there is significant historical human consumption of PLRV and associated obligatory proteins in multiple potato varieties and locations (Powell and Mondor, 1976; Thomas, 1983). A conservative estimate of current consumer consumption of PLRV replicase protein is to assume that a third of all potato tubers produced in the U.S. have detectable levels of PLRV virus and therefore, the presence of PLRV replicase can be assumed. Although infection reduces the yield and quality of potatoes, humans and animals have safely consumed such potatoes for centuries and food experts consider these infected potatoes as generally recognized as safe.

nptII gene and NPTII protein The nptII gene and its encoded NPTII protein is used to select for transformed plant cells during the plant transformation process. In general, the frequency of cells that are transformed is often as low as 1 in 10,000 or 1 in 100,000 of the cells treated. Therefore, to facilitate the selection of transformed cells, a selectable marker gene coupled with a selective agent incorporated into the growing media for treated plant cells is employed. Consequently, cells selected for plant regeneration contain the selectable marker gene nptII as well as CPB and PLRV resistance genes. The NPTII enzyme encoded by the nptII gene uses ATP to phosphorylate neomycin and the related kanamycin, thereby inactivating these antibiotics in

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the selective cell growing media, preventing them from killing the cells. The sole purpose of inserting the nptII gene into potato cells is to have an effective method of selecting plant cells that contain the introduced genes. The safety assessment for the NPTII plant marker was reviewed by Fuchs et al., (1993b). This marker has been extensively used for numerous crops and a history of safe use has been established.

Safety Assessment of NewLeaf Plus Potato Tubers Food and feed safety evaluations of the tubers produced by NewLeaf potato plants took into consideration the safety of the expressed proteins and assessment of the composition and wholesomeness of the tubers produced by the transformed plants. On the basis of results derived from studies performed to evaluate the integrity of the T-DNA insert, it was concluded that no proteins other than the PLRVrep, Cry3A and NPTII proteins could be produced from the inserted transgenes. The proteins produced by the genes (cry3A and nptII) have been thoroughly tested for potential mammalian toxicity and have a history of safe human consumption. The PLRVrep protein is not produced at detectable levels in potato plants. The allergenic potential of the Cry3A and NPTII, proteins was assessed and found to be of no concern (Lavrik et al., 1995) Both Cry3A and NPTII are rapidly digested in gastric fluids and do not contain amino acid sequences matching those of known allergens or toxins (Betz et al. 2000). It is well known that PLRV proteins and plant virus replicase proteins are routinely consumed without deleterious health effects (EPA 1997). PLRV replicase is not considered to be an allergen nor does it have any amino acid sequence homology to known allergens.

Characterization of inserted DNA Characterization of inserted DNA was carried out on the isolated genomic DNA to determine the actual plasmid DNA transferred into the plant genome. The characterization used standard molecular techniques of polymerase chain reaction (PCR) and Southern blot analyses. The analysis defined the integrity of genetic elements that were transferred from the plasmids to the genome of the potato lines and the linkage between the three genetic elements (PLRVrep, cry3A, and nptII). Additionally, the presence of PLRVrep mRNA and Cry3A and NPTII proteins was determined by molecular and biochemical methods. Growth of cultured plant cells on selective media and resistance of field grown plants to the target pests demonstrated that the expression cassettes were intact and functional (Thomas and Kaniewski, 1998; Rogan et al., submitted). Furthermore, the stability of the genetic material in the genome is ensured as each selection is propagated via tuber based cloning and not through the set of viable seed. Over four years of field tests across the potato production areas of North America confirmed that the viral and insect resistance genes consistently function as expected. Potato plants grown in multiple year/site field tests showed a high level of resistance to PLRV and CPB when natural levels of exposure to these pests vary from low to high (as determined from incidence in control plots), thus supporting the stability of these genes under the environmental conditions of production agriculture (Duncan et al. 1999: Thomas et al., 2000; Rogan et al., submitted).

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Gene expression in NewLeaf Plus potato plants and tubers Protein characterization studies were conducted to establish that Cry3A and NPTII proteins are produced in NewLeaf Plus potato lines and are equivalent to the previously characterized reference standards, and to those produced in previously deregulated and approved potato plant lines. The results from these analysis confirmed that the Cry3A and NPTII proteins produced in NewLeaf Plus potato lines are functionally and chemically equivalent to Cry3A and NPTII proteins produced in bacterial expression systems. Protein levels for Cry3A and NPTII were estimated in potato tissue extracts using enzyme linked immunosorbent assay (ELISA), which is a highly specific assay developed and validated to estimate the concentration of the respective protein in extracts derived from plant tissues. Expressed Cry3A protein levels correspond to a range of 7.84 – 12.50 µg/g leaf tissue while average levels in tubers ranged from 0.24 – 0.44 µg/g tuber tissue on a fresh weight basis (Table 1). The expression level of NPTII protein in leaf and tuber tissue was below the detection limits of the assay (0.3 µg/g tissue fresh weight). The PLRV replicase protein was below the detection limits of a highly sensitive immunoassay. It is likely that the protein is highly unstable due to proteolytic degradation (Van der Wilk et al., 1997). Therefore, to demonstrate that the PLRVrep gene was functional, messenger RNA (mRNA) analyses were conducted using Northern and RNA dot blotting techniques. The Northern blot demonstrated that mRNA of the expected size was produced from the inserted transgene (Lawson et al., 2001). The results of a RNA dot blot test showed that the amount of PLRV viral RNA in naturally infected conventional Russet Burbank potato plants was 5 to 10 fold higher than the transgene-derived mRNA expressed in leaf and tuber tissues of NewLeaf Plus potato plants.

Safety of the introduced Cry3A Protein safety studies were conducted to assess the safety of the Cry3A and NPTII proteins. The safety of NPTII protein was addressed in publications by Fuchs et al. (1993a, 1993b), Flavell et al. (1992), Nap et al. (1992) and Betz et al. (2000). Therefore, this assessment will focus only on the Cry3A protein. As part of the safety assessment of the Cry3A protein, it was important to consider that the Cry3A protein has an established history of safe use. The safety assessment of the Cry3A protein focused on two key topics: allergy and toxicology. Data are summarized below to address these two topics.

History of safe use of Cry3A The Cry3A protein introduced into the Russet Burbank has a long history of safe use. The Cry3A protein expressed in NewLeaf Plus potato plants is indistinguishable from the Cry3A protein contained in microbial formulations that have been used safely commercially for the past six years (EPA 2000; Lüthy et al., 1982). These microbial formulations have been used on a wide variety of crops, including fresh produce like eggplant, potato and tomato, with no reported allergenic or toxic responses, establishing a basis for the lack of allergenic or toxic concern for the Cry3A protein.

Allergenicity of Cry3A Large quantities of a vast variety of proteins are consumed in human diets each day from all foods. Rarely do any of these tens of thousands of proteins elicit an allergenic response

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(Taylor, et al.,1992). Although there are no predictive assays available to definitively predict the allergenic potential of proteins (Food and Drug Administration, 1992), the history of safe use and the biochemical profile of the Cry3A protein provides a basis for allergenic assessment when compared with known protein allergens. Allergenic proteins are often, though not always, glycosylated proteins. Protein allergens must be stable to the peptic and tryptic digestion and the acid conditions of the digestive system if they are to reach and pass through the intestinal mucosa to elicit an allergenic response (Astwood et al., 1996). Another significant factor contributing to the allergenicity of proteins is their high concentration in foods that elicit an allergenic response (Taylor, 1992; Taylor et al. 1987; and Taylor et al., 1992). The Cry3A protein does not possess any of those characteristics common to protein allergens (Lavrik et al., 1995; Betz et al., 2000). The history of safe use provides an additional level of assurance. More importantly, the Cry3A protein was shown to be very labile to digestion by proteases which mimic those present in the mammalian gastric digestive system, minimizing the potential for this protein to be absorbed by the intestinal mucosa. In vitro, simulated mammalian gastric and intestinal systems digestive mixtures were established and used to assess the susceptibility of Cry3A protein to proteolytic digestion. The method of preparation of the simulated digestion solutions used is described in The United States Pharmacopeia (1989), a frequently cited reference for in vitro digestion. In vitro studies with simulated digestive solutions are widely used as models of animal digestion. They have been used to investigate the digestibility of plant proteins (Nielson, 1988; Marquez and Lajolo, 1981), animal proteins (Zikakis et al., 1977) and food additives (Tilch and Elias, 1984); to assess protein quality (Akeson and Stahmann, 1964); to study digestion in pigs and poultry (Fuller, 1991); to measure tablet dissolution rates to monitor biodegradation for pharmaceutical applications (Alam et al., 1980); and to investigate the controlled-release of experimental pharmaceuticals (Doherty et al., 1991). The data from the simulated digestion experiments demonstrated a half-life for the Cry3A protein of less than 30 seconds in the gastric system (Betz et al., 2000). As expected, in the intestinal system, the 68,000 dalton Cry3A protein was rapidly converted to a trypsin-resistant core of approximately 55,000 daltons, which was not further degraded. To put the rapid degradation of the Cry3A protein in the simulated gastric system into perspective, solid food has been estimated to empty from the human stomach by about 50% in two hours, while liquid empties 50% in approximately 25 minutes (Sleisenger and Fordtran, 1989). Therefore, any Cry3A protein remaining after cooking would be rapidly degraded in the gastric system upon consumption. Since most protein allergens are glycosylated, the Cry3A protein, as purified from the tuber of CPB resistant potato, was examined for glycosylation and was shown not to be glycosylated (EPA, 1995). This result was expected since enzymatic glycosylation in plants requires passage through the rough endoplasmic reticulum and Golgi bodies (Taiz and Zeiger, 1991). This transport requires specific targeting sequences on the protein, which were not engineered into the Cry3A gene and hence the resulting Cry3A protein. The Cry3A protein contained no targeting sequence. Because there is apparently no glycosylation of the Cry3A protein, it does not fit the profile of most common allergenic proteins. The Cry3A protein shows no significant homology to any known protein allergen including any of the 121 amino acid sequences reported for allergens (using allergen as the key word) in the three current protein databases (Genpept, Pir protein and Swissprot databases) (Betz et

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al., 2000). There was no greater homology of the native Cry3A protein to any of the 121 amino acid sequences for the allergenic proteins than for a scrambled sequence of the same amino acids that comprise the Cry3A protein. Finally, most allergens are present as major protein components in the specific food. This is true for the allergens in milk (Baldo, 1984; Lebenthal, 1975; Taylor, 1986; Taylor et al., 1987), soybean (Shibasaki et al., 1980; Burks et al., 1988; Pedersen and Djurtoft, 1989), peanuts (Barnett et al., 1983; Sachs et al., 1981; Barnett and Howden, 1986; Kemp et al., 1985), etc. In contrast to this generality for common allergenic proteins, the Cry3A protein is present in potato tubers at very low levels, up to approximately 0.0002% of fresh weight of the potato tuber and approximately 0.01% of the total protein (Lavrik et al., 1995; Betz et al., 2000). The low levels of the Cry3A protein in potato tubers and digestive labiality of this protein relative to that for known food allergens establishes an extremely low probability that the Cry3A protein will be consumed, absorbed via the intestinal mucosa during consumption and trigger production of IgE antibodies responsible for allergenicity. Acute oral gavage of Cry3A An acute oral gavage study was conducted in mice to confirm the mammalian safety of the Cry3A protein. Following EPA guidelines, a high dose of the test substance (5000 mg of Cry3A protein per kg body weight) was administered to the test animals. The dose was higher than that expected to be consumed in the diet by over 2.5 million times based on the average human consumption of potatoes and the level of the Cry3A protein present in the tuber (Lavrik et al., 1995). No adverse effects were observed in test animals and there were no effects on food consumption, weight gain or gross pathology (Lavrik et al., 1995; Betz et al., 2000). In summary, the data and analyses described above support the conclusion that there is no reason to believe that the Cry3A protein should pose any significant allergenic risks for consumption of the products generated from NewLeaf Plus potato plants. The lack of activity in the acute gavage study, no sequence homology to known toxins and allergens, and an established history of safe use in microbial formulations, support the safety of the Cry3A protein for human or animal consumption. Safety of the introduced PLRVrep protein It is well known that PLRV proteins and plant virus replicase proteins are routinely consumed without deleterious health effects (EPA 1997). EPA has concluded that genes, DNA and RNA themselves are not a safety concern. PLRV replicase is not considered to be an allergen nor does it have any amino acid sequence homology to known allergens. PLRVrep protein was never detected in NewLeaf Plus potato plants, so a study of the levels of mRNA was conducted. The results of a RNA dot blot test showed that the amount of PLRV viral RNA in naturally infected conventional Russet Burbank potato plants, tubers of which are routinely consumed in the human diet, was 5 to 10 fold higher than the transgene-derived mRNA expressed in leaf and tuber tissues of NewLeaf Plus potato plants. Compositional Analysis and Nutritional Assessment of NewLeaf Potatoes Compositional data on the NewLeaf Plus Russet Burbank tubers was generated on the basis of scientific recommendations for establishing the substantial equivalence of transgenic crops compared to conventional varieties, for the purpose of safety and nutritional assessment. International expert groups from the Organization for Economic Cooperation and Development (OECD), Food and Agricultural Organization of the United Nations (FAO)

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and the World Health Organization (WHO) concluded that compositional analysis of the new food source should be conducted and the results compared to an appropriate counterpart which has an accepted standard of safety (FAO/WHO, 1991; OECD, 1993; WHO, 1995; FAO, 1996). Experts consistently have recommended that this compositional comparison be based on key nutrients and toxicants or anti-nutrients for the food source in question. Key nutrients have a substantial impact in the overall diet and include major constituents such as fats, proteins and carbohydrates and minor components such as certain minerals and vitamins. Key toxicants are those toxicologically significant components known to be present in the species whose toxic potency and level may be significant to animal or human health. In potatoes, these are anti-nutrients called steroidal glycoalkaloids that are found in most Solanum species. They have been documented as the cause of human poisonings (Zitnak, 1977). Because glycoalkaloids are a distinct threat to human health, analyses for their concentrations are conducted on new potato varieties. The key constituents in potatoes appropriate for compositional analysis to establish substantial equivalence are glycoalkaloids, total solids, sugars (reducing or dextrose and sucrose), protein and vitamin C (Love, 2000). Analyses were conducted on field harvested NewLeaf tubers to assess the levels of these key nutrients and anti-nutrients in potato tubers to assess whether there were any changes in these components compared to tubers of conventional potato varieties, including the parental controls from which NewLeaf Plus Russet Burbank was generated. Field studies necessary to generate the data package were conducted over a period of four years in all of the major potato growing regions of North America (Rogan et al. 2000). All field sites were implemented and managed according to regulations in the country where conducted (United States or Canada). Compositional studies of the potato tubers derived from the NewLeaf Plus line showed that the nutritional properties of the potato tubers were substantially equivalent to those of conventional Russet Burbank and similar white potato varieties and that the NewLeaf Plus tubers have normal low levels of naturally occurring anti-nutrients (Table 2). The compositional analysis conducted on tuber samples from field sites across North America showed that the potato tubers from the commercialized NewLeaf Plus lines are substantially equivalent to tubers derived from control potatoes of the same variety and conventional potatoes presently in commerce (Love, 2000; Rogan et al. 2000). Key potato constituents (total solids, sugars, vitamin C, protein, natural glycoalkaloid toxicants) and proximate analysis (ash, moisture, calories) were well within the expected range for commercially produced potato tubers as reported in the scientific literature. The nutritional properties of the NewLeaf Plus potato tubers are equivalent to those from potatoes already in commerce (Rogan et al. 2000). Table 2 summarizes the compositional and nutritional analysis for NewLeaf Russet Burbank potatoes compared to control potatoes grown in the same fields and to the expected range for these nutrients as reported in the scientific literature for potatoes introduced through traditional breeding programs. The results of compositional analysis confirm that genetic improvement, through Agrobacterium mediated transformation, of NewLeaf Plus potatoes did not alter nutrient composition from that of conventionally bred potatoes. As a result of the analyses of molecular characterization, protein safety and expression and nutrient composition, it was concluded that tubers produced by NewLeaf Plus potato plants are as safe for consumption as conventional potatoes. Use of these potato tubers in 1998 through 2000 for fresh market and processing further confirmed these potatoes show no differences in taste or suitability for

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processing. Uses included fresh market and processed potato products (frozen and dehydrated primarily).

Environmental Safety Assessment Environmental risk assessments for biotech plant introductions center on whether or not a trait introduced into the crop variety is capable of making a crop that was not a weed into a weed, whether the trait can be transmitted to wild relatives and thereby make them more weed like or whether the trait makes the transformed plants more susceptible to pests and diseases. Additionally, the impact of the environmental introduction of the plant on non-target organisms, such as beneficial insects and wildlife, needs to be assessed. Field work was supplemented by specific laboratory studies to determine if the Cry3A protein poses a risk to non-target organisms. Laboratory studies combined with field observations of beneficial insect populations are used. Prior to commercial introduction of NewLeaf Plus potato plants, field trials with NewLeaf Plus lines were conducted at multiple locations throughout the potato growing regions of North America. Some field trials were conducted to select lines that were agronomically equivalent to conventional potato varieties. For safety assessments, lines were also evaluated on the weediness potential in accordance with USDA guidelines. Three years of agronomic performance data from field trials showed these potato plants performed no differently than conventional potato plants. NewLeaf Plus potatoes conformed to the expected disease and insect resistances of the parental variety Russet Burbank. The genes introduced into NewLeaf Plus Russet Burbank have demonstrated no ability on their own to induce disease nor do plants containing these introduced genes show increased susceptibility to the common potato disease and insect pests including other plant viruses, fungal blights, aphids, spidermites and leaf hoppers. Overall there was no significant difference in agronomic performance and weediness potential between commercialized NewLeaf Plus potato plants and conventional potato plants of the same varieties except in regard to the target pests PLRV and CPB (Lavrik et al., 1995) Volunteer potatoes can easily be controlled with good field sanitation and management practices, including the use of herbicides and tillage. There are no native species within the potato production areas of North America that can naturally cross with NewLeaf plants to produce viable seed (www.aphis.usda.gov/biotech/potato.html; OECD 1997). Therefore, the probability of vertical gene flow is vanishingly low. There is no meaningful potential for the potatoes themselves to become weeds (EPA, 2001). The risks to non-target organisms, including earthworms, beneficial insects and small mammals, were accessed to be insignificant for commercial production of NewLeaf Plus potatoes relative to standard potato varieties. The Cry3A protein is non-toxic to tested animals (Table 3). The non-target insect species included larvae honey bee (Apis mellifera L.), a beneficial insect pollinator; green lacewing larvae (Chrysopa carnea), a beneficial predatory insect; parasitic wasps (Brachymeria intermedia), a beneficial parasite of the housefly; the ladybird beetle (Hippodamia convergens), a beneficial predaceous insect; and earthworms (Eisenia fetida). The US EPA determined that the Cry3A protein is nontoxic to non-target mammalian species based on their review of toxicity studies in mice (EPA, 2001). Exposure of parasitic wasps, ladybird beetles, green lacewings and honey bee larvae to purified Cry3A protein by ingestion demonstrated no toxicity at a maximum hazard dose of 100 ppm (Betz et

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al., 2000; as cited in EPA 2001). Earthworms were also unaffected at soil-incorporated levels 100 times the concentration of Cry3A protein estimated to be present in soil of NewLeaf potato fields (100 mg a.i./kg dry soil) (as cited in EPA, 2001). Two species of the common soil insect Collembola were also tested and Cry3A protein was not toxic at a level >200ppm (as cited in EPA, 2001). The US EPA has determined that the planting of NewLeaf potatoes will not affect any threatened or endangered species (EPA, 2001). Field studies conducted to evaluate the efficacy of Cry3A protein expressed in potato plants and conventional spray programs against CPB demonstrated that this protein expression technology as first demonstrated in NewLeaf potatoes was highly effective in suppressing CPB populations (Perlak et al., 1993). NewLeaf potato plants were not defoliated by CPB whereas plants receiving no control measure for CPB were nearly 100% defoliated before reaching maturity. Field observations of NewLeaf Plus Russet Burbank confirmed the findings in this variety (Rogan et al., submitted). The PLRV protein has no insecticidal properties and specifically impacts only the potato leafroll virus. Since PLRV is ubiquitous in the environment, wildlife and arthropods are exposed to the PLRVrep gene and replicase protein in standard Russet Burbank fields with no observed deleterious effects. Studies conducted in Washington and Idaho on the populations of beneficial arthropods in NewLeaf Plus potato fields compared to those in standard Russet Burbank fields consistently demonstrated that populations are higher in NewLeaf Plus fields (Thomas et al., 2000; Betz et al. 2000). The beneficial arthropods monitored included ladybird beetles, predatory true bugs, lacewings, spiders and parasitic wasps. However, plants improved with viral gene sequence could potentially pose a unique hypothetical concern in regards to creation of new plant virus types. One of the theoretical risks associated with the use of viral transgenes concerns the possibility that an invading virus may recombine with transgene mRNA and create a more virulent recombinant form of the original virus (Allison et al., 1996). Several investigators have demonstrated that viral transgenes can recombine (Lommel and Xiong, 1991 and Gal et al., 1992). However these laboratory-based experiments were done under extreme selection conditions and high innoculum pressure where constructs were specifically designed to select for very rare recombinant strains of the virus.

On the basis of theoretical and empirical evidence, the likelihood that recombinant viruses will arise due to the commercial production of NewLeaf Plus Russet Burbank is remote. Genetically modified NewLeaf Plus potato plants were modified with the nature identical PLRVrep gene (orf1/orf2) derived from PLRV. This gene contains no significant sequence homology with viral genes other than those found in different PLRV strains and has no available replicase binding regions. Therefore, it is expected not to be capable of recombination. Furthermore, observations (seed certification records, natural viral inoculation and intentional passage of PLRV through NewLeaf Plus plants using spreader rows) taken during extensive field tests with NewLeaf Plus potatoes at numerous locations and over several years have established that NewLeaf Plus potato plants have not generated more virulent (recombinant) plant viruses (Thomas et al., 1998). Even if recombination did occur it would not result in a more virulent virus as neither the PLRV replicase gene nor its gene products are known to contribute to traits that increase virus virulence such as cell-to-cell movement, systemic plant infection and vector transmission.

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Insect Resistance Management for Cry3A Because the Colorado potato beetle has a history of developing resistance to chemical pesticides, an insect resistance management program was implemented to support the commercial introduction of NewLeaf potatoes. Growers limited planting of NewLeaf Plus potatoes to no more than 80% of their potato acres. The remaining acres, planted to conventional, non-Bt potato varieties, acts as a “refuge” reducing the possibility that CPB resistance to the Cry3A protein will develop. In a total of 5 years of monitoring for resistance in potato fields planted to NewLeaf and NewLeaf Plus varieties, there have been no confirmed reports of CPB surviving in either NewLeaf or NewLeaf Plus fields.

Conclusion NewLeaf Plus potatoes contribute value to potato producers through reduction in the amount of pesticides needed to prevent damage from PLRV and CPB. In 1998, the Reibe and Zalewski (in press) found that NewLeaf Plus potato use reduced insecticide and miticide applications by 80% over that for conventional farmed Russet Burbank in the Columbia Basin growing region. This represents a potential elimination of a significant portion of the 500,000 kg of insecticide active ingredient applied to the 35,000 ha of Russet Burbank grown in the Columbia Basin alone. Individual NewLeaf Plus growers realized an average net savings of approximately $331/ha because of the reduction in pesticide applications and the higher quality of the harvested tuber due to the reduction in net necrosis. The reduction in tuber net necrosis in NewLeaf Plus compared to Russet Burbank was worth an average of $302/ha alone in sales returns to the grower. Monsanto Company developed NewLeaf Plus potato that is resistant to infection by potato leafroll virus and to feeding by the Colorado potato beetle. Genes isolated from naturally occurring sources were inserted into the genome of Russet Burbank potatoes to produce potato plants resistant to these major plant pests. The NewLeaf Plus Russet Burbank selection was commercialized following safety reviews by three United States regulatory agencies (United States Department of Agriculture, United States Environmental Protection Agency and the Food and Drug Administration). Two Canadian regulatory agencies (Canadian Food Inspection Agency and Health Canada) and two Japanese agencies (Ministry of Health, Labor and Welfare and Ministry of Agriculture, Food and Fisheries) subsequently conducted their own individual reviews of the submitted safety package. Data generated on the genes, gene products, agronomic properties, and tuber nutrient composition support the finding that NewLeaf Plus potatoes are no different from conventional Russet Burbank potatoes except for the introduced traits. For food and feed, these data support the finding that the tubers from these potatoes are as wholesome as conventional potatoes in commerce. Information and data contained within this document have been provided to regulatory authorities for review. Regulatory review continues as we update regulatory files and make submissions to additional countries globally.

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Table 1. Levels (micrograms/gram tuber fresh weight) of Transgene Expression Products in Tubers and Leafs Derived from NewLeaf Plus Potato Plants.

Clone Number Tissue Type Variety Cry3Aa NPTII RBMT21-350 Tuber NL Plus Russet

Burbank 0.34 (0.24-0.44)b

<0.0027

RBMT21-350 Leaf NL Plus Russet Burbank

10.09 (7.84 – 12.50)b

<0.0027

a Numbers in parenthesis represent minimum and maximum levels for the Cry3A proteins in leaf and tuber tissue. Leaf samples were collected at approximately six to nine weeks post-planting; tuber samples were collected at harvest. b Samples were harvested from 1996 field trials conducted at 3 sites in Canada (New DenMark, New Brunswick, Spruce Grove, Alberta and Winkler, Manitoba). Plants were grown using a randomized complete block design with four replicates per clone. Means were derived from analyses of tubers obtained from 4 plots per site.

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Table 2. Proximate Analysis of Tubers from NewLeaf Plus Russet Burbank and Russet Burbank Control (1995-1996 Field Trials)

Component Russet Burbank

NewLeaf Plus Russet Burbank

Literature range/mean value

Characteristics† Mean (Range)‡ Mean (Range)‡ Literature Reference

Protein % 4.95 1 4.3-5.1 4.85 1 4.5-5.3 3.4-7.3 Pavek, et al. 1980-1992

Ash % 4.75 2 4.48-4.96 4.7 2 4.64-4.82 2.2-9.5 Approximate value from Burton 1989, Kadam et al. 1991 and Scherz and Sensor 1989

Total Solids % FW 21.6 1 20.7-22.9 21.75 1 20.9-22.6 16.8-24.5 Pavek, et al. 1980-1992 Calories/100g 381.8 2 380.9-383.2 382.1 2 381.3-382.7 350 Average value from Burton, Kadem et al. 1991,

Scherz and Sensor 1989 Moisture g/100g 1.48 2 1.15-1.84 1.43 2 1.18-1.59 NA Sucrose % FW 0.177 1 0.129-0.236 0.172 1 0.124-0.239 0.10-0.88 Results are average of tubers from eleven individual

trials at Aberdeen, ID Dextrose % FW 0.167 1 0.072-0.356 0.154 1 0.064-0.342 0.04-0.53 Results are average of tubers from eleven individual

trials at Aberdeen, ID Total Carbohydrates

g/100g 85.18 2 83.54-86.89 85.17 2 82.66-86.87 84.5 Average Value from Burton 1989, Kadem et al. 1991,

Schertz and Sensor 1989 Vitamin C

mg/100 FW 14.1 1 9.1-17.2 13.35 1 8.7-18.3 10.3-22.0 Results are average of tubers from eleven individual

trials at Aberdeen, ID Glycoalkoids mg/100g FW

6.3 1 3.8-14.9 6.5 1 3.3-14.4 3.1-16.1 Pavek, et al. 1980-1992

† Protein, fat, ash, carbohydrate, moisture and calories reported based on dry weight of sample; sugars, glycoalkaloids, vitamin C reported based on fresh weight. ‡ Range denotes the lowest and highest individual values across sites for each cultivar or event. 1 Value reported is mean of combined data from 6 sites, each with 4 replicates, across the potato growing regions of the United States and Canada. 2 Value reported is mean of combined data from 3 sites in Canada, each with 4 replicates.

August 2002 25

Table 3: Toxicity of Cry3A Protein to Non-target Organisms. Results of Laboratory Studies.

Non target Organism Test results & findings

Reference

Lady Bird Beetle NOEL1 >100ppm As cited in EPA 2001

Collembola NOEL >200ppm Betz et al. 2000

Honey Bee Larva NOEL >100ppm As cited in EPA 2001

Parasitic Wasp NOEL >100ppm As cited in EPA 2001

Green Lacewing Larva NOEL >100ppm As cited in EPA 2001

Earthworms NOEL >100mg/kg dry soil As cited in EPA 2001

Acute oral toxicity mouse NOEL>5220mg/kg/day Betz et al. 2000

1 No Observable Effect Level