1

Introduction and Background

1.1 THE IMPORTANCE OF CROP PROTECTION

Farmers have been trying to minimize the impacts of crop pests for thousands of years. Insects, nematodes, bacteria, fungi, and viruses can cause massive destruction of important crops, and this destruction can have great socioeconomic effects. For example, the Irish potato famine of the 1800s led to the deaths of about 1 million people and large-scale emigration. More recently, head blight caused by the fungal pathogens Fusarium graminearum and F. poae caused about $3 billion in damage to wheat and barley in 1991-1996 (US Wheat and Barley Scab Initiative 1998). An estimated $7 billion in crop losses per year in the United States are caused by nematodes (NSTC 1995) and even greater losses are caused by arthropod pests. In addition to socioeconomic effects, some plant pests pose human health hazards, such as those caused by fungal mycotoxins. Pest control is a continuous process: as pest-protected plants are bred or new chemical pesticides are developed, pests evolve to overcome these control methods.

Early methods to control pests include the use of sulfur fumigation in 1000 BC, ants for biocontrol in 324 BC, and crop rotation, controlled irrigation, and manure application during the Roman Empire. Arsenic was used in the 1600s, Bacillus thuringiensis was developed as a microbial insecticide as early as 1938 (NRC 1996), and the use of synthetic pesticides became the predominant means of pest control in the 1940s. Since the 1960s, there has been wide implementation of integrated pest-management (IPM) approaches, designed to use a variety of natural controls and



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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION 1 Introduction and Background 1.1 THE IMPORTANCE OF CROP PROTECTION Farmers have been trying to minimize the impacts of crop pests for thousands of years. Insects, nematodes, bacteria, fungi, and viruses can cause massive destruction of important crops, and this destruction can have great socioeconomic effects. For example, the Irish potato famine of the 1800s led to the deaths of about 1 million people and large-scale emigration. More recently, head blight caused by the fungal pathogens Fusarium graminearum and F. poae caused about $3 billion in damage to wheat and barley in 1991-1996 (US Wheat and Barley Scab Initiative 1998). An estimated $7 billion in crop losses per year in the United States are caused by nematodes (NSTC 1995) and even greater losses are caused by arthropod pests. In addition to socioeconomic effects, some plant pests pose human health hazards, such as those caused by fungal mycotoxins. Pest control is a continuous process: as pest-protected plants are bred or new chemical pesticides are developed, pests evolve to overcome these control methods. Early methods to control pests include the use of sulfur fumigation in 1000 BC, ants for biocontrol in 324 BC, and crop rotation, controlled irrigation, and manure application during the Roman Empire. Arsenic was used in the 1600s, Bacillus thuringiensis was developed as a microbial insecticide as early as 1938 (NRC 1996), and the use of synthetic pesticides became the predominant means of pest control in the 1940s. Since the 1960s, there has been wide implementation of integrated pest-management (IPM) approaches, designed to use a variety of natural controls and

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION cultural methods to suppress pest populations (Smith and Van denBosch 1967). IPM is an approach which manages pests by biologically integrated alternatives for pest control (US Congress 1947, as amended in the 1972 Federal Environmental Pesticide Control Act, section 136r(a)) and is “a sustainable approach to managing pests by combining biological, cultural, physical, and chemical tools in a way that minimizes economic, health, and environmental risks” (US Congress 1947, as amended by the 1996 Food Quality Protection Act, section 136r-1). Pesticides are used only as necessary and when other control methods have failed (Stern et al. 1959). 1.2 DIVERSE GENETIC MODIFICATION METHODS To develop pest-resistant or tolerant cultivars, plant breeders have taken advantage of natural genetic variation or induced mutations. The methods that plant breeders use depend on the type of cultivar they want to improve (for example, an inbred line, a hybrid, or a population) and the reproductive biology of the plant (for example, self-pollinated or cross-pollinated) (Fehr 1987; Stoskopf et al. 1993). An inbred line (or purebred) is phenotypically uniform1, and the progeny2 are identical with the parent. Many self-pollinated crops are released as inbred lines (for example, soybeans, Glycine max, and barley, Hordeum vulgare). A hybrid is the cross between two or more inbred lines; it can also be phenotypically uniform but not genetically identical with the parents. Many cross-pollinated crops are released as hybrids (for example, corn or maize, Zea mays). A plant population results from crossing a number of lines and is genetically and phenotypically diverse, although for key traits, a population can be phenotypically uniform (for example, every plant resistant to a pest). All genetic modification methods for crop improvement consist of introducing variation, selecting useful variants, and field-testing the selected lines, hybrids, or populations to determine their merit. In the past, almost all commonly used plant breeding techniques began with artificial crosses, in which pollen from one plant is transferred to a reproductive organ of another, sexually compatible plant. Crossing allows for the combining of desirable traits, such as pest resistance and increased yield, from two or more plant cultivars.3 The objective is to combine these traits in a new cultivar that is superior to its parents. To overcome some of the 1   Expressing the same phenotypes or traits. 2   Offspring. 3   Cultivated variety of plant.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION barriers to sexual hybridization between cultivated and wild relatives, rescue of pollinated embryos has been used: when a cross yields a viable embryo but the surrounding seed endosperm4 is not viable, the embryo is taken from the nonviable seed environment and “rescued” by being grown in tissue culture. Other techniques to introduce variation in cultivars include cell fusion, somaclonal variation, chemical or x-ray mutagenesis, and genetic engineering (see section 2.4.2). Cell fusion is used to produce novel combinations of genomic material from nuclei and organelles when plants are not sexually compatible (Ehlenfeldt and Helgeson 1987); it can be performed only on plants that can be cultured with protoplast technologies. With protoplast technologies, cells are disconnected from tissues, their walls are removed, and their membranes are prepared for fusion. Somaclonal variation is variation that occurs during the tissue-culture process, and its phenotypic outcomes are often similar to other forms of mutagenesis. Genetic engineering is the transfer of a or a few genes into a cultivar with the use of Agrobacterium tumefaciens, microprojectile bombardment, electroporation, or microinjection. Transgenic methods will be discussed in more detail in subsequent sections of this report. One of the main differences among the techniques used for introducing variation is in the amount of DNA involved. In progeny resulting from a cross between two cultivars, half the genome comes from each cultivar. Each half (haploid) genome contains a significant amount of DNA (table 1.1). The amount transferred with conventional breeding in the case of Arabidopsis could be 70 megabases (Mb) (half the progeny's haploid genome comes from each parent). For bread wheat, the amount of DNA could be almost 8000 Mb. In contrast, transgenic methods involve the addition of only a few genes and flanking regulatory sequences (totaling about 1-20 kilobases). Another important difference among the techniques can be the source of the transferred DNA. Sexual hybridization involves genes from sexually compatible species, which tend to be rather similar. Mutagenesis and the somaclonal variation process do not add genes, but rather modify existing genes. Cell fusion can add genes from evolutionarily divergent plant species (such as, plants from different genera), but normally fused cells are from somewhat related plants (for example, the technique has not been conducted by fusing cells from plants and microorganisms). In genetic engineering or transgenic methods, genes from any organism in the biosphere can be used as long as the regulatory sequences are functional in the host plant. For example with genetic engineering researchers 4   Nutritional tissue in seeds.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION TABLE 1.1 Genome Size of Common Plants     Nuclear DNA Content Common Name Scientific Name Picograms (diploid nucleus)a ~Millions of base pairs (haploid nucleus)b Arabidopsis Arabidopsis thaliana 0.3 145 Barley Hordeum vulgare 10.1 4873 Brussels sprout Brassica oleracea ssp. gemmifera 1.3 628 Corn Zea mays 4.75-5.63 2292-2716 Cotton Gossypium hirsutum 4.39 2118 Oats Avena sativa 23.45 11315 Papaya Carica papaya 0.77 372 Peanut Arachis hypogeae 5.83 2813 Rice Oryza sativa 0.87-0.96 419-463 Soybean Glycine max 2.31 1115 Tobacco Nicotiana tabacum 8.75-9.63 4221-4646 Tomato Lycopersicum esculentum 1.88-2.07 907-1000 Bread Wheat Triticum aestivum 33.09 15966 Wild Wheat Triticum monococcum 11.92 5751 a 1 picogram = 965 million base pairs, haploid nucleus b DNA content of unreplicated haploid chromosome complement Source: Data from Arumuganathan and Earle, 1991. have added genes to potatoes from bacteria, viruses, chickens, and moths. The foreign gene can also be modified by molecular techniques before introduction into the plant (for example, by incorporating DNA base pair substitutions). However, a key question is whether the fact that genes can be obtained from broader sources for plant biotechnology inherently impacts the safety of the resulting genetically engineered organism (see section 2.2.1 and section 2.4.2). Foreign genes engineered into plants may or may not be homologous to genes already present in the plant or the food supply. 1.3 HISTORY AND IMPACT OF BREEDING METHODS Selection for desirable traits and hybridization has been used since the advent of human agriculture, but the logic underlying the inheritance of traits was not discovered until the middle 1800s. In the 1860s, Gregor Mendel demonstrated the process of heredity by hybridizing different varieties of pea (Pisum sativum) and examining traits such as flower and seed color, seed and pod shape, flower position, and plant height in sub-

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION sequent generations. His revolutionary experiments paved the way for modern agriculture by showing that through controlled pollination crosses, characteristics are inherited in a logical and predictable manner. In 1905, Roland Biffen, of England, built on Mendel's experiments by illustrating that the ability of wheat (Triticum aestivum) to resist a rust fungus could be passed to later generations (NAS 1998). Since then, many plants have been bred to include desirable traits, such as pest resistance. Blight resistance traits from a Mexican potato species (Solanum demissum) have been introduced into over 50% of all potato cultivars (NRC 1989). Blight-resistant corn (Zea mays), rust-resistant wheat (T. aestivum), and aphid-resistant alfalfa (Medicago sativa) are other notable examples of conventional plant breeding. Major gains in crop yields have been attributed partially to advances in classical plant breeding and plants developed for pest resistance. Corn yields have increased from 5 metric tons per hectare in 1967 to 8 metric tons per hectare in 1997, cereal harvests have been increasing at an average rate of 1.3% per year (Mann 1999), world food production has doubled since 1960, and agricultural productivity from land and water use has tripled (NSTC 1995). Conventional breeding will likely continue to play an essential role in the improvement of agricultural crops. However, many believe that traditional breeding methods will not be sufficient to meet increasing demands in developing countries for staple crops, such as wheat ( T. aestivum), rice (Oryza sativa) and corn (Zea mays) (Mann 1999). Classical methods are time-consuming (that is they take approximately 10 years to develop a variety) and labor-intensive (only one line of thousands becomes a useful variety). In addition, beneficial traits can be linked to or lead to undesirable traits, such as disease susceptibility. For example, when male-sterile corn was extensively grown in the 1960-1970s to promote hybrid-corn production, a new race of southern corn leaf blight fungus (Helminthosporium maydis) evolved which successfully attacked this type of corn and significantly decreased US corn yields (Dewey et al. 1988). Some have proposed transgenic methods to augment the advances in conventional breeding. 1.4 EMERGENCE OF RECOMBINANT DNA AND OVERVIEW OF TRANSGENIC PEST-PROTECTED PLANTS In the past two decades, scientists have focused on expanding genetic modification methods to include the use of recombinant DNA (rDNA) techniques. New varieties generally can be produced faster by rDNA than by conventional breeding methods. rDNA methods allow the introduction of genes from distantly related species or even from different

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION biologic kingdoms. In addition, detailed knowledge of the trait being introduced (such as a DNA sequence or cellular function) can lead to less variability in the offspring and eliminate some of the uncertainty about linked traits. The site of insertion of a gene can affect its expression, but generally plants with the appropriate level of expression can be selected if a number of transgenic plants are produced. After a trait is introduced by transgenic methods, the resulting plant can be sexually hybridized with useful varieties developed by conventional breeding. 1.4.1 Emergence of Recombinant DNA Methods Recombinant DNA methods emerged in the early 1970s after the discovery of restriction enzymes (Linn and Arber 1968; Meselson and Yuan 1968), DNA-sequencing methods (Sanger and Coulson 1975; Maxam and Gilbert 1977), and plasmid and viral vectors for engineering organisms (Jackson et al. 1972;Cohen et al. 1973). The methods have been used ever since to manipulate DNA fragments that contain genes of interest. With the advent of this technology, concerns about the safety of experiments that use rDNA methods developed. The National Academy of Sciences (NAS) in 1974 convened a committee to assess the safety concerns associated with rDNA research. The committee recommended that rDNA experiments be postponed until further evaluation of the risks (Berg et al. 1974). Soon after, the International Conference on Recombinant DNA Molecules, better known as the Asilomar Conference, was held (Berg et al. 1975). An outline of guiding principles and restrictions for rDNA research was generated at this conference. In 1976, the principles were reviewed by the National Institutes of Health (NIH), which implemented official guidelines to be administered by the NIH Recombinant DNA Advisory Committee (RAC) (NIH 1976). The guidelines focused on laboratory containment of rDNA microorganisms. As more experiments were conducted and more data on the risks were generated, less restrictive guidelines were put into place (NIH 1978). In recent years, the guidelines have been expanded to include other rDNA applications, such as gene therapy, and have been adopted not only by institutions receiving federal funding, but also by industry and state institutions. About a decade after the emergence of rDNA technology, genes from the bacterium Agrobacterium tumefaciens were used to carry foreign genes into plants. Agrobacterium inserts portions of its tumor-inducing (Ti) plasmid-encoded genes into plant chromosomes as part of its natural, parasitic life cycle (Nester et al. 1983). When researchers add foreign DNA (such as a gene for pest-protection) in between Ti plasmid-encoded insertion sequences, the foreign DNA sequences are also inserted into the plant's chromosome. The first transgenic plants were developed with

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION Agrobacterium-transformation methods (Horsch et al. 1985). Since then, other methods for plant transformation, such as electroporation and particle-gun transformation, have been developed (Klein et al. 1987; Finer et al. 1999); these methods allow transformation of plants that are not natural hosts for Agrobacterium. 1.4.2 Development of a Regulatory Framework for Transgenic Plants Concurrently with developments in the technical aspects of genetically engineering crops by using rDNA methods, regulatory concerns about the release of genetically engineered organisms into the environment emerged. The NIH guidelines in 1978 prohibited the environmental release of genetically engineered organisms unless exempted by the NIH director. In 1982, the RAC reviewed a request to field test “ice-minus” bacteria, strains of Pseudomonas syringae and Erwinia herbicola that had inactivated ice-nucleation genes (Lindow and Panopoulos 1988). NIH approved the request in 1983 (NIH 1983). The approval of the field trial was controversial and sparked several court cases that invoked the National Environmental Policy Act (NEPA) (US Congress 1969). NEPA requires that any agency decision that significantly affects the quality of the environment be accompanied by a detailed statement or an assessment of the environmental impacts of the proposed action and of alternatives to it. As the field trial was being debated by the courts, a congressional hearing was held at which questions were raised about the ability of federal agencies to address hazards to ecosystems in light of the uncertainties (US Congress 1983). At a second hearing in 1984, the Senate Committee on Environment and Public Works discussed the potential risks with representatives of the Environmental Protection Agency (EPA), NIH, and the US Department of Agriculture (USDA). The government agencies stated that existing statutes were sufficient to address the environmental effects of genetically engineered organisms (US Senate 1984). Also in 1984, a White House committee was formed under the auspices of the Office of Science and Technology Policy (OSTP) to propose a plan for regulating biotechnology. In 1986, OSTP published the Coordinated Framework for the Regulation of Biotechnology (OSTP 1986), which is still used today. The framework is based on the principle that techniques of biotechnology are not inherently risky and that biotechnology should not be regulated as a process, but rather that the products of biotechnology should be regulated in the same way as products of other technologies. The coordinated framework outlined the roles and policies of the federal agencies and contained the following ideas: existing laws were, for the most part, adequate for oversight of biotechnology products; the products, not the process, would

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION be regulated; and genetically engineered organisms are not fundamentally different from nonmodified organisms. A 1987 National Academy of Sciences white paper came to similar conclusions, recommending regulation of the product, not the process, and stating that genetically engineered organisms posed no new kinds of risks, that the risks were “the same in kind” as those presented by nongenetically engineered organisms (NAS 1987) (section 2.2.1). The coordinated framework considered existing regulations and laws that were potentially applicable to biotechnology and proposed how EPA, USDA, and the Food and Drug Administration (FDA) would cooperate to review the safety of biotechnology products. USDA was designated the lead agency for regulating genetically engineered crops. FDA was designated to review transgenic crop varieties used for food under the Federal Food, Drug, and Cosmetic Act (FFDCA; US Congress 1958). EPA later clarified its interpretation of the statutes to include “plant pesticides” for regulation under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA; US Congress 1947) and FFDCA (EPA 1994a, c). NEPA is applicable to all federal agencies. The current regulation of biotechnology products is shown in table 1.2. Not all the products in table 1.2, namely plant-pesticides, were discussed in the original coordinated framework. Shortly after the coordinated framework was developed, USDA reviewed and approved transgenic crop varieties for field trials under the Federal Plant Pest Act (FPPA;US Congress 1957).5 According to the act, a plant pest is: any living stage of … insects, mites, nematodes, slugs, snails, protozoa, or other invertebrate animals, bacteria, fungi, other parasitic plants or reproductive parts thereof, viruses, or any organisms similar to or allied with any of the foregoing, or any infectious substances, which can directly or indirectly injure or cause disease or damage in any plants or parts thereof, or any processed, manufactured, or other products of plants. Using the coordinated framework as a guide, USDA was the first agency to propose a regulation for the review of plants genetically modified with rDNA methods. On June 16, 1987, a Federal Register notice established procedures for obtaining permits for releasing genetically engineered organisms into the environment in field trials (USDA 1987). Under that regulation, coverage extends to organisms or substances that meet the definition of a plant pest or that USDA chooses to designate as a plant pest. 5   The FPPA supplements and extends the much older Plant Quarantine Act.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION TABLE 1.2 Summary of the Current Regulation of Biotechnology Products, as currently described on the USDA website for Regulatory Oversight in Biotechnology Agency Jurisdiction Laws US Department of Agriculture Plant pests, plants, veterinary biologics Federal Plant Pest Act Food and Drug Administration Food, feed, food additives, veterinary drugs, human drugs, medical devices Federal Food, Drug, and Cosmetic Act EPA Microbial and plant-pesticides, new uses of existing pesticides, novel microorganisms Federal Insecticide, Fungicide, and Rodenticide Act; Federal Food, Drug, and Cosmetic Act; Toxic Substances Control Act Source: USDA 1999e. 1.4.3 The First Field Trials In November and December 1987, USDA issued permits for three engineered herbicide-tolerant varieties of tomato (two from DuPont and one from Calgene) and two herbicide-tolerant varieties of tobacco (from Calgene). These plants were tolerant of the herbicides glyphosate, bromoxynil, or sulfonylurea. Tolerance was based on the overexpression of the herbicide target in the tolerant plant, the expression of resistant forms of the target enzymes, or the expression of enzymes that could degrade the herbicide (Comai et al. 1985; Harrison et al. 1996). Transgenic pest-protected plants were developed in parallel to herbicide-tolerant plants. The first transgenic pest-protected plant was engineered to contain a coat-protein gene from the tobacco mosaic virus (TMV) (Powell-Abel et al. 1986); the gene confers resistance to TMV itself, and to viruses similar to TMV. A transgenic TMV-resistant tomato line developed by Monsanto was approved for field trials on March 23, 1988. Today, a large portion of US corn and cotton acreage is planted with transgenic pest-protected plants (Economic Research Service 1999a, b; Carozzi and Koziel 1997). Those transgenic pest-protected plants contain genes from the bacterium Bacillus thuringiensis (Bt). Bt produces several proteins during sporulation including endotoxins. Upon ingestion by an insect, the protoxin form of endotoxin undergoes cleavage in the insect gut to a truncated active form, which kills insects by binding to receptors

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION in the insect gut and forming pores (Gill et al. 1992). The pores cause the gut contents to leak into the blood, and this eventually leads to insect death. About 60 proteins from more than 50 subspecies of Bt have been identified in the last 20 years (Federici 1998). Which insects Bt toxins affect depends on the class of Bt protein; they include moths and butterflies (lepidopterans), flies and mosquitoes (dipterans), and beetles (coleopterans). Mixtures of Bt have been used to spray crops for over 50 years. However, the Bt toxin generally loses its effectiveness in the environment within a few days. Sometimes spraying needs to be done frequently. In transgenic crops, Bt toxin is continuously produced and is protected from the elements. It therefore retains its ability to kill pests during the entire growing season. Moreover, the toxin is generally expressed in every part of the plant, including internal tissues that are difficult to protect with topically applied pesticides. This internal production provides protection against pests that are internal feeders such as the pink bollworm in cotton and the European corn borer in corn. On the other hand, the constant presence of Bt toxin in transgenic pest-protected plants during the growing season has led to concerns about its persistence in the environment and increased probability of pest evolution to overcome the protection mechanism. The first permit to field-test transgenic crops that contained Bt genes was issued to Monsanto in 1988 for tomato. Initial attempts to make crops that would resist pests in the field were not successful because of problems with the expression of the bacterial genes. In 1990, the first successful Bt crop, cotton (Gossypium hirsutum), was produced by overcoming translational and transcriptional barriers to bacterial-gene expression in plants (Perlak et al. 1990). Transgenic methods for introducing Bt are often followed by conventional breeding with varieties that express other useful agronomic traits. Before transgenic crops were commercialized (from 1987 to 1994), the USDA approved field trials of nine nematode-resistant transgenic pest-protected plant varieties, 45 fungus-resistant varieties, 17 bacteria-resistant varieties, 322 insect-resistant varieties, and 194 virus-resistant varieties. 1.5 AGENCY POLICIES REGARDING COMMERCIALIZATION OF TRANSGENIC PEST-PROTECTED PLANTS 1.5.1 USDA Policy In 1992, four years after the first field trials began, USDA proposed a regulation that described a petition process for determining that particular plants would no longer be regulated and therefore could be commer-

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION cially planted. The regulation was finalized in 1993 (USDA 1993). For a crop to achieve nonregulated status, “environmental assessment ” and “determination of nonregulated status” documents are prepared by USDA; the documents address safety concerns under the FPPA such as impacts on agriculturally beneficial organisms, as well as addressing the agency's NEPA requirements (section 4.1.1). 1.5.2 FDA Policy Also in 1992, FDA published a policy statement on its role under FFDCA for reviewing new plant varieties developed by all methods, whether transgenic or conventional (FDA 1992) (section 4.1.2). The FFDCA authorizes FDA to control foods that are “adulterated” with added substances, including naturally occurring substances. The 1992 policy established FDA's role in reviewing the overall composition of the nutrients and toxicants in genetically modified plants. The policy states that “key factors in reviewing safety concerns should be the characteristics of the food product, rather than the fact that the new methods are used.” Under the 1992 policy, FDA asks that companies develop information to determine whether or not the company is obligated to come to the agency for formal regulatory review. Considerations include genetic stability, compositional and nutritional quality attributes of the plant, and toxicity and allergenicity of the gene product. FDA requires that companies submit nutritional and safety data to the agency if there is reason to believe that new plant varieties may pose risks. After publishing its 1992 policy, FDA recommended that companies developing transgenic varieties consult with the agency before marketing a new variety. Guidelines for this voluntary consultation process were published by the FDA in October 1997 (FDA 1997c). Over 45 transgenic plants, including numerous transgenic pest-protected plants and all crop plants that have been marketed in the United States, have gone through the consultation process. FDA has not required that any of the proteins added to transgenic plants be reviewed as food additives 6. 1.5.3 EPA Policy In the early 1990s, EPA held numerous public meetings of the Biotechnology Science Advisory Committee (BSAC) to develop its regulations for products of biotechnology. In 1994, EPA published proposed 6   However, the antibiotic resistance genes in the Flavr Savr tomato were reviewed as food additives at the request of the tomato's manufacturer (section 1.5.4).

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION rules that help clarify the agency's role in the coordinated framework by describing which plant-pesticides would be regulated under FIFRA and FFDCA and which would be exempt from regulation (section 4.1.3). Although the proposal has not been finalized, the agency has been implementing its essential elements in registering or exempting plant-pesticides since 1995. EPA defined a plant-pesticide as “a pesticidal substance produced in a living plant and the genetic material necessary for the production of that pesticidal substance, where the substance is intended for use in the living plant.” The genetic material necessary for production of a pesticidal substance was included in the definition of plant-pesticide to enable regulatory coverage, under FIFRA, of plant parts such as seeds and pollen where the pesticidal substance might not be expressed. However, with regard to regulation under FFDCA, the agency proposed to establish a categorical exemption from the requirement of a tolerance for this genetic material. EPA's proposed FIFRA regulation establishes three categories of plant-pesticides that would be exempt from regulation under FIFRA. The first category contains plant-pesticides whose genetic material encodes for a pesticidal substance that is derived from plants that are sexually compatible. The second category of plant-pesticides that are exempt from FIFRA regulation are those that act by affecting the plant so that the target pest is inhibited from attaching to and/or invading the plant tissue by, for example, acting as a structural barrier, or by inactivating toxins produced by the target pest. The third category consists of substances that are coat proteins of plant viruses. Substances that are exempt from regulation under FIFRA are not automatically exempt from the requirement of a tolerance under FFDCA. Therefore, EPA proposed three additional regulations under FFDCA to accomplish this. Similar to the FIFRA exemption, pesticidal substances derived from plants that are sexually compatible are proposed to be exempt from the requirement for a tolerance. In addition, pesticidal substances derived from plants that are not sexually compatible would also be exempt from the requirement of a tolerance provided that the following two conditions are met: (1) the genetic material encoding the pesticidal substance is derived from a food plant; and (2) the pesticidal substance does not result in a new or significantly different human dietary exposure. EPA also proposed to exempt coat proteins of plant viruses and, as noted earlier, to exempt the genetic material that encodes pesticidal substances. In 1997, EPA published supplemental notices of proposed rulemaking for the FIFRA and FFDCA proposals published in 1994. EPA took this action in order to allow the public to comment on the Agency's evaluation of the requirements imposed by the Food Quality Protection Act of 1996

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION (FQPA) (Public Law 104-170, EPA 1997b) that the agency did not address in the 1994 proposals. FQPA amended FFDCA and FIFRA to include a new safety standard for pesticide residues on food, one notable change being special safety factors for children. 1.5.4 Commercialization of Transgenic Pest-Protected Plants Under the above USDA, FDA, and EPA statutes, the first transgenic crop varieties were approved for commercial planting in the early 1990s. In 1992, the first transgenic crop variety achieved nonregulated status from USDA. This variety was a tomato line for altered fruit ripening developed by Calgene (Flavr Savr). In addition to USDA review, FDA reviewed the safety and nutritional aspects of the Flavr Savr tomato and a food additive petition from Calgene for the use of the kanamycin resistance trait in tomatoes, cotton, and canola (Kahl 1994). Since this review, FDA conducts its assessments for genetically engineered crops by consulting with companies about the safety and composition of the variety and has not required a food additive petition for any other transgenic product, although it could make such a request in the future. EPA was not involved in reviewing the Flavr Savr tomato because the transgenic modification of the tomato did not involve a pesticidal trait. In December 1994, the first transgenic pest-protected plant achieved nonregulated status from USDA: a virus-resistant squash variety developed by Upjohn/Asgrow Seed Company that contained watermelon mosaic virus-2 coat protein and zucchini yellow mosaic virus coat protein. The USDA assessments for this crop address such concerns as the likelihood of creating new plant viruses via recombination of the introduced coat-protein gene with naturally occurring viruses, the potential of the two new virus-resistance genes to cause squash to become a weed, and the movement of the genes to wild squash relatives. EPA also reviewed this crop. In the July 27, 1994, Federal Register, EPA published a notice that Asgrow Seed Co. had submitted a pesticide petition to EPA under FFDCA to exempt the coat proteins from the requirement of a tolerance (EPA 1994b). EPA reviewed the petition for safety concerns, such as toxicity, and established an exemption from the requirement of a tolerance under FFDCA for “residues of the plant-pesticides, as expressed in Asgrow line ZW20 of Cucurbita pepo L. and the genetic material necessary for the production of these proteins.” EPA also proposed to exempt viral coat protein genes and gene products from review and registration under FIFRA (Section 1.5.3) (EPA 1994a and 1997b). Varieties employing the Bt resistance mechanism were the next pest-protected plants to achieve nonregulated status from USDA and to have their gene products reviewed as plant-pesticides by EPA. A Bt potato line

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION TABLE 1.3 Plant Pesticides Reviewed by EPA Protein Source Cry9Ca Bacillus thuringiensis Cry1A(b)b Bacillus thuringiensis Cry1A(c)b Bacillus thuringiensis Cry3Ac Bacillus thuringiensis viral coat protein cucumber mosaic virus viral coat protein papaya ringspot virus viral coat protein virus Y viral coat protein watermelon mosaic virus viral coat protein zucchini yellow mosaic virus replicase potato leaf roll virus a Reviewed for use in corn. b Reviewed for use in all plants. c Reviewed for use in potatoes. Source: EPA 1999a resistant to beetles was developed by Monsanto and was cleared for commercial release by USDA in March 1995, subject to EPA and FDA review. The Cry3A delta-endotoxin from Bt was reviewed by EPA in early 1995. An exemption under FFDCA from the requirement of a tolerance for this Bt toxin and the genetic material necessary for its production eliminated the need to establish a maximal permissible level for residues of this Bt toxin in potatoes. For the exemption, EPA reviewed data on toxicity and allergenicity and convened a subpanel of the FIFRA Scientific Advisory Panel to discuss its review; the panel concluded that the Bt potato presented “little potential for human dietary toxicity.” Table 1.3 lists the plant pesticides that have been reviewed by EPA. 1.5.5 Current Profile of Transgenic Plants Over 40 transgenic crop varieties have been cleared through the federal review processes for commercial use in the United States. Of them, 17 (as of December 1999) contain transgenes for pest-protection. Of the 17, 14 containing Bt genes have been developed and cleared by USDA for commercial release (table 1.4) (USDA 1999b). Although the EPA 1994 rule is not yet final, the plant-pesticides in these crops have been reviewed and their gene products registered as plant-pesticides by EPA (table 1.3). Five virus-resistant transgenic pest-protected plant varieties have achieved nonregulated status from USDA (table 1.4). Transgenic crops were first planted commercially in the 1995 growing season. Since then, their use has been rapidly increasing. In 1997, 20.3

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION TABLE 1.4 Crops Deregulated by the USDA with Transgenic Pesticidal Traits Crop Resistancea Company Date of Nonregulated Status Insect resistance Potato Bt IR Monsanto March 1995 Corn Bt IR Ciba-Geigy May 1995 Cotton Bt IR Monsanto June 1995 Corn Bt IR Monsanto August 1995 Corn Bt IR Northrup King January 1996 Corn Bt IR Monsanto March 1996 Potato Bt IR Monsanto May 1996 Corn Bt IR Dekalb Genetics March 1997 Cotton Bt IR, HR Calgene April 1997 Corn Bt IR, HR Monsanto May 1997 Tomato Bt IR Monsanto March 1998 Corn Bt IR, HR AgrEvo May 1998 Virus and insect resistance Potato Bt IR, VR Monsanto December 1998 Potato Bt IR, VR Monsanto February 1999 Virus resistance only Squash VR Upjohn/Asgrow December 1994 Squash VR Asgrow June 1996 Papaya VR Cornell University September 1996 aBt IR = Bt endotoxin-based insect resistance; VR = virus resistance; HR = herbicide resistance Source: USDA 1999b. million acres of transgenic crops were planted in the United States; in 1998, 50.2 million acres were planted (James 1998); and in 1999, 70 million were planted (James 1999). A total of 98 million acres were planted worldwide in 1999 (James 1999). Transgenic pest-protected crop varieties that contain Bt toxin transgenes make up a large percentage of the commercial transgenic crops. In 1998 in the United States, about 25% of total cotton acreage and 21% of total corn acreage were planted with transgenic crops that contain Bt genes (USDA 1999d). In 1999, approximately 30 million acres of insect protected crops were planted in the US (James 1999). So far, many of these transgenic pest-protected crops seem to be effective in controlling pests; a reduced need for chemical pesticides and increased yields have been reported by many, but not all, growers (Robinson 1998; Gianessi and Carpenter 1999). For example, one report indicates that insecticide sprays in cotton were reduced in 1998 from an average of 8.3 insecticide applications for conventional cotton to an average of 6.0 sprays

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION for Bt cotton (Mullin and Mills 1999), which led to an estimated reduction of over 5 million acre-treatments and over 2 million pounds of chemical insecticide (Gianessi and Carpenter 1999). However, estimated benefits might depend on the baseline level of pest infestation during a specific growing season and on the techniques used to make comparisons (USDA 1999d). The use of transgenic pest-protected crops has been profitable in growing regions subject to severe pressure from specific pests or where alternative means of pest control have been infeasible or expensive. For example, Bt cotton has been accepted by a large percentage of growers in states where pest resistance to synthetic pyrethroids has left them without chemical means of controlling bollworms, but limited in other regions where pest hazards are not so extreme (USDA 1999d; Falck-Zepeda et al. 1999). Adoption of Bt corn has similarly been limited to areas with the highest pest pressure (USDA 1999d). In addition to the approved commercial transgenic crop varieties, thousands of transgenic varieties are undergoing field trials (USDA 1999c). From 1987 through January 2000, the number of permits issued and notifications acknowledged was over 6700; about 3000 were for varieties having pest-resistance genes (table 1.5). 1.6 PUBLIC CONCERNS AND ISSUES Given the rapid increase in acres planted with commercial transgenic crops and the likely additional increase in their use, many groups have raised concerns about the ecological and human health risks that might be posed by these crops (Ho 1998). Although the risks might not, in principle, differ in type from those associated with other products (for example, conventional pest-protected plants, pesticides), the public has focused its attention on transgenic crops. Concerns over pesticidal traits include the enhanced evolution of resistant pest strains, the toxicity or allergenicity of the gene products to TABLE 1.5 Number of Permits Issued for or Notifications of Field Trials in the United States Involving Crops with Pest-Resistance Genes, 1987-1999. Category of Resistance Number (% of all field trials) Insect 1505 (22) Virus 1013 (15) Fungal 378 (5.5) Bacterial 78 (1.2) Nematode 7 (0.1)

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION humans, the hybridization of transgenic pest-protected plants with neighboring wild relatives, and adverse effects on nontarget organisms. These concerns are presented below and discussed more extensively in chapter 2 and chapter 3 where the scientific bases and empirical evidence are analyzed. 1.6.1 The Development of Pest Resistance to Engineered Traits Farmers and gardeners who use microbial Bt sprays are concerned that the widespread commercial planting of transgenic pest-protected plants with Bt genes will lead to rapid development of insect resistance to Bt, which will in turn make their microbial sprays ineffective. Instances of pest adaptation to conventional Bt products have been documented (Tabashnik et al. 1994). Scientists who conduct research on pest resistance to plant-protection mechanisms published resistance management strategies for Bt corn, cotton, and potato (McGaughey and Whalon 1992; Tabashnki 1994; Roush 1997; Gould 1998; UCS 1998), and the EPA published findings of a specially convened scientific advisory panel on Bt resistance management (SAP 1998). Under the registration process for plant pesticides, EPA requires a particular amount of non-Bt cotton or corn to be planted next to Bt cotton or corn to serve as a refuge for insects carrying Bt susceptible genes, and they also encourage the development of resistance management strategies for other transgenic Bt crops. However, the percentage of acreage that is needed to provide a sufficient refuge to avoid the rapid development of pest resistance and the proper location of the refuge are debated by industry, entomologists, and environmental groups (Inside EPA 1999; UCS 1998) (see section 2.9). Recently, the EPA placed new restrictions on growing transgenic Bt corn which include a requirement that farmers plant 20% to 50% of their corn acreage with conventionally bred corn (EPA 1999h; Weiss 2000). 1.6.2 Human Health Concerns Allergenicity due to transgenic gene products has been highlighted as a human health concern (Metcalfe et al. 1996a, b) (see section 2.5.1). Guidance for assessing these concerns was provided in a 1996 report published by the International Food Biotechnology Council in conjunction with the International Life Sciences Institute (Metcalfe et al. 1996b). One transgenic plant was shown to have allergenic properties during laboratory tests (Nordlee et al. 1996). To improve the nutritional quality of soybeans, a transgenic plant containing a methionine-rich protein from Brazil nuts (Bertholletia excelsa) was developed by Pioneer Hybrid International. The company discontinued development of this product as a result of these

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION allergenicity concerns. It is important to note that modern biotechnology can also be used to reduce the allergenic risks associated with our current food supply. For example, Matsuda et al. (1998) have published papers showing that they reduced the major allergen in rice by approximately 80% by using antisense rDNA technology. 1.6.3 Gene Flow and Cross Pollination With Weedy Relatives Other safety issues which have received attention are those involving ecological risks such as the effects of gene flow. Studies have been conducted to assess the potential for gene flow among and within related species (see section 2.7 and section 3.4.1). The ability of transgenic plants to cross-pollinate with their wild relatives and form offspring with enhanced weediness has been investigated when herbicide-tolerant rapeseed plants were back-crossed with a wild relative. The hybrid progeny plants produced an equivalent amount of seed as the wild genotypes and were also herbicide-resistant (Snow et al. 1999). That study indicated that back-crossed generations of hybrids between transgenic and nontransgenic crops can have the same potential to flourish as other plants. In a more controversial study, wild type Arabidopsis thaliana plants were found to be fertilized by pollen from transgenic plants more often than by pollen from nontransgenic plants (Bergelson et al. 1998). In addition to those experiments, the use of models has been explored to assess the invasiveness of engineered organisms, although indications are that these models will require several years worth of data to be validated (Kareiva et al. 1996). 1.6.4 Nontarget Species Although some transgenic pest-protected plants have the potential to reduce pesticide use and thus to prevent substantial environmental damage, there is concern that gene products from the plants could harm beneficial insects or birds (nontarget species) that are in direct contact with the plants or that feed on insects that are (see section 2.6 and section 3.1.2). Hillbeck et al. (1998a, b) found that when chrysopid larvae were reared on prey that were fed Bt-producing corn, they had 62% mortality. When they were reared on prey that were fed non-Bt corn, mortality was only 37%. Another experiment indicated that Bt toxins can bind to humic acids from soil, be protected from biodegradation, and persist in the soil (Crecchio and Stotzky 1998). It is not known whether nontarget organisms would be affected by bound toxin molecules in field situations. Other studies indicate that Bt toxins generally degrade quickly in the soil (Palm et al. 1994; Sims and Sanders 1995; Palm et al 1996). A well-publicized recent laboratory study indicated that when mon-

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION arch butterfly larvae were fed milkweed dusted with transgenic Bt pollen, high mortality was exhibited (Losey et al. 1999). The relationship between this preliminary laboratory finding and field effects is unclear (Yoon 1999). One recent field test reports that at least 500 pollen grains per square centimeter is necessary to sicken monarch caterpillars and that milkweed plants growing adjacent to corn fields had only an average of 78 grains per square centimeter (Kendall 1999) (see section 2.6.2). In other experiments, however, monarch caterpillars that consumed concentrations of Bt corn pollen (Event 176) naturally deposited on milkweeds in the field experienced 20% mortality with only 48 hours of exposure (Hansen and Obrycki 1999a,b). Further field-based research is needed to determine whether dispersed Bt pollen could have detectable effects on the population dynamics of nontarget organisms. 1.6.5 Regulatory Concerns The above concerns have led some to question the safety review that transgenic crops receive in the United States under the coordinated framework. Many believe that transgenic crops present substantial human health and ecological risks, and that these are not properly assessed by the regulatory framework. But many others believe that the risks are minimal, that the benefits outweigh the risks, and that the current regulatory scheme is perhaps onerous. Cited benefits include a reported 250,000-gallon reduction in chemical pesticide use in 1996 and a 30-50% reduction in the number of insecticide applications over the period of 1996-1998 due to the growing of commercial transgenic Bt cotton (Robinson 1998; Williams 1997, 1998, and 1999). The reduction might prevent much environmental damage. In addition, Bt toxins have specific insect targets, whereas traditional broad-spectrum chemical insecticides often kill insects more indiscriminately (Federici 1998). This may lead to outbreaks of secondary pests requiring the use of more insecticides. However, many believe that transgenic pest-protected plants should not only be compared to the use of chemicals, but also to alternative methods such as biological control. The debate has intensified in recent months, given the international concerns and impending regulatory decisions in the United States. In March 1999, Congress held a hearing on the 1994 proposed EPA plant-pesticide rule (Hart 1999c). Although transgenic pest-protected plants have been registered under this rule in the last 5 years, the rule has not been finalized, and its scientific and legal validity are being questioned. The EPA planned to finalize the rule by the end of 1999. The debate over this rule has many facets. Environmental and consumer groups argue that the EPA is not rigorous enough in its scientific review (Hart 1999c)

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION and that the proposed rule has too many exemptions. They are also concerned that the EPA rule does not adequately cover all of the risk issues. Several professional societies have argued that EPA is overstepping its boundaries by reviewing plant gene products as pesticides, stating that this could damage the progress of science by overburdening small biotechnology companies and public breeding programs with the cost of regulation, as well as undermining confidence in the food supply (Eleven Scientific Societies 1996; CAST 1998). Some congressional members are concerned about the lack of a formal cost-benefit analysis to accompany the rule and about whether the definition of a pesticide in FIFRA gives EPA the authority to regulate transgenic pest-protected plants (Hart 1999c). Given the debate about its proposed rule, EPA held a workshop in 1997 to address some of the criticisms (EPA 1997c) and is incorporating changes into the rule on the basis of comments. One comment that is being considered suggests changing the terminology to avoid the use of “plant-pesticides” for gene products of transgenic pest-protected plants. EPA has sought input on a more appropriate name for these traits in a recent Federal Register notice (EPA 1999c). A change might address the public's concern about labeling plants as “pesticides”; however, it would not address other concerns, such as EPA's authority, its role in the coordinated framework, and whether the risks are being properly addressed by this framework. 1.7 ROLE OF THIS REPORT In the past, the National Academy of Sciences (NAS) and National Research Council (NRC) have had the opportunity to provide guidance to scientists, regulatory agencies, and the public concerning rDNA issues. The 1974-1975 efforts helped to initiate the national debate over the safety of genetically engineered organisms (Berg et al. 1974). In 1987, given the proposed release of genetically engineered organisms into the environment, the NAS Council issued a white paper, Introduction of Recombinant DNA-Engineered Organisms into the Environment (NAS 1987), which proposed guiding principles that helped shape national policy for the review of genetically engineered organisms. In 1989, the NRC convened a committee to establish a framework for decisions regarding the field testing of genetically engineered organisms (NRC 1989); the criteria and methods for evaluation suggested by that committee have been guiding USDA oversight of field trials for transgenic crops in the last 10 years. Given the current political and social climate, the NRC believes that it has a role to play in addressing the scientific issues surrounding the regulation of transgenic pest-protected plants.

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GENETICALLY MODIFIED PEST-PROTECTED PLANTS: SCIENCE AND REGULATION The scope of the study and structure of this report are outlined in the executive summary (section ES.1). Transgenic pest-protected plants7 are the focus of the committee's discussion of the regulatory framework, inasmuch as the framework is designed for transgenic plants (OSTP 1986). Conventional pest-protected plants are discussed for scientific comparisons. Given impending decisions with respect to the EPA plant-pesticide rule, the committee focused on the EPA's proposed rule, but also addressed the roles of the EPA, USDA, and FDA under the coordinated framework. The committee hopes that this report will provide guidance for reviewing the thousand or more transgenic pest-protected plants that are being tested in the field as well as those yet to be developed. Although transgenic Bt crops have received the most attention given their commercial use, the committee proposes to look towards the future by discussing general issues concerning transgenic pest-protected plants for which there may be fewer data and that could have an impact in coming years. It is not possible for the current committee to comment on other classes of transgenic crops (such as herbicide-tolerant crops) given the breadth of the issues and the time frame; however, some of the conclusions in this report regarding transgenic pest-protected plants might be applicable to other transgenic crops and are indicated as such. Terms that frequently appear in the report are defined in the executive summary (ES.3), a list of acronyms can be found in appendix D, and common and scientific names for the various organisms listed in the text appear in appendix E. 7   Note that the committee focused on potential impacts of food and fiber crops, not on the potential impacts of other types of transgenic pest-protected plants that might be commercialized in the future (for example, forest trees).