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APHIS Regulatory Policy for Transgenic Organisms

OVERVIEW

This chapter reviews the regulations and procedures currently used to guide the Animal and Plant Health Inspection Service (APHIS) in overseeing the environmental safety of testing and commercialization of transgenic plants. It also describes the historical development of these regulations and procedures. The broad overview provided here leads into Chapter 4, which presents case studies of APHIS oversight of specific transgenic crops.

Chapter 1 described the U.S. Coordinated Framework for the Regulation of Biotechnology created in 1986 for the regulation of development and commercialization of transgenic organisms in the United States and Chapter 2 described the scope and regulatory triggers that can be used as clarified in 1992 (OSTP 1992). Except for pesticidal plants, USDA-APHIS-BBEP (Biotechnology, Biologics, and Environmental Protection) has primary regulatory authority for environmental assessment of all transgenic plants. This chapter specifically examines the oversight of transgenic plants by APHIS under the Federal Plant Pest Act (FPPA) and the Federal Plant Quarantine Act (FPQA). The final chapter of this report considers how that oversight might change under the Federal Plant Protection Act of 2000.

The scope of APHIS’s review includes, under the FPQA, importation of regulated articles and interstate movement of regulated articles and, under the FPPA, environmental release of regulated articles. “Regulated article” is defined below. “Environmental release” is defined as “the use



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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation 3 APHIS Regulatory Policy for Transgenic Organisms OVERVIEW This chapter reviews the regulations and procedures currently used to guide the Animal and Plant Health Inspection Service (APHIS) in overseeing the environmental safety of testing and commercialization of transgenic plants. It also describes the historical development of these regulations and procedures. The broad overview provided here leads into Chapter 4, which presents case studies of APHIS oversight of specific transgenic crops. Chapter 1 described the U.S. Coordinated Framework for the Regulation of Biotechnology created in 1986 for the regulation of development and commercialization of transgenic organisms in the United States and Chapter 2 described the scope and regulatory triggers that can be used as clarified in 1992 (OSTP 1992). Except for pesticidal plants, USDA-APHIS-BBEP (Biotechnology, Biologics, and Environmental Protection) has primary regulatory authority for environmental assessment of all transgenic plants. This chapter specifically examines the oversight of transgenic plants by APHIS under the Federal Plant Pest Act (FPPA) and the Federal Plant Quarantine Act (FPQA). The final chapter of this report considers how that oversight might change under the Federal Plant Protection Act of 2000. The scope of APHIS’s review includes, under the FPQA, importation of regulated articles and interstate movement of regulated articles and, under the FPPA, environmental release of regulated articles. “Regulated article” is defined below. “Environmental release” is defined as “the use

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation of a regulated article outside the constraints of physical containment that are found in a laboratory, contained greenhouse, or a fermenter or other contained structure” (APHIS 1987). For example, growing regulated articles in a controlled greenhouse would not constitute a release, but growing them in a standard greenhouse would. When a regulated article is determined to have “nonregulated status,” it no longer is subject to APHIS oversight, but the agency has the option of taking action on any plant that it believes to present a plant pest risk as long as they have not determined it to have nonregulated status. In this case the article has been evaluated, and determined with a reasonable degree of certainty not to present risk as a plant pest, and APHIS cannot regulate it under FPPA or FPQA. In 1987 APHIS published regulations for the release of transgenic organisms into the environment (APHIS 1987). These were modified in 1988 and 1990 to exempt Escherichia coli strain K-12, sterile strains of Saccharomyces cerevisiae, asporogenic strains of Bacillus subtilis, and Arabidopsis thaliana from permitting requirements for interstate movement under specified conditions (APHIS 1988, 1990) and again in 1993 and 1997 to provide a notification procedure and a process to petition for nonregulated status (APHIS 1993, 1997a). The rules and their revisions are promulgated under the statutory authority of the FPPA and FPQA, which grant APHIS broad powers to regulate potential plant pest risks. APHIS must grant a permit for the introduction of any regulated article. Although there are several fine distinctions in its definition (see BOX 3.1), a regulated article is a genetically engineered organism that is or contains genetic material from one of many taxa listed in 7 CFR 340.2 or that meets the definition of a potential plant pest. The definition of “plant pest” is discussed in more detail below. Anyone wishing to “introduce” such an organism must request a permit from APHIS. APHIS has streamlined the permitting process for most transgenic plant species under specified conditions; for these, formal notification of APHIS in advance of a release is often sufficient for obtaining permission to introduce the transgenic plant. In addition, APHIS has developed a process whereby an applicant can petition the agency to determine that a transgenic organism does not pose a plant pest risk and is therefore no longer a regulated article subject to APHIS regulatory oversight. The coordinated framework explicitly provides that federal agencies should focus on the characteristics of risks posed by a biotechnology product, not on the process by which it is created, but APHIS uses the process of genetic engineering to trigger oversight. As discussed in Chapter 2, APHIS has argued that it was not treating genetically engineered organisms differently from so-called established plant pests or naturally occur-

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation BOX 3.1 Key Definitions Used by APHIS Introduce or introduction (APHIS 1997b) “To move into or through the United States, to release into the environment, to move interstate, or any attempt thereat.” Plant (APHIS 1987) “Any living stage or form of any member of the plant kingdom including, but not limited to, eukayotic algae, mosses, club mosses, ferns, angiosperms, gymnosperms, and lichens (which contain algae) including any parts (e.g., pollen, seeds, cells, tubers, stems) thereof, and any cellular components (e.g., plasmids, ribosomes, etc.) thereof.” Plant pest (APHIS 1987) “Any living stage (including active and dormant forms) 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 agents or substances, which can directly or indirectly injure or cause disease or damage in or to any plants or parts thereof, or any processed, manufactured, or other products of plants.” Regulated article (APHIS 1987) “Any organism which has been altered or produced through genetic engineering, if the donor organism, recipient organism, or vector or vector agent belongs to any genus or taxon designated in a list (7 CFR 340.2) of taxa known to have plant pests, and meets the definition of plant pest, or is an unclassified organism and/or an organism whose classification is unknown, or any product which contains such an organism, or any other organism or product altered or produced through genetic engineering which the Director, BBEP (Biotechnology, Biologics, and Environmental Protection division of APHIS), determines is a plant pest or has reason to believe is a plant pest. Excluded are recipient microorganisms which are not plant pests and which have resulted from the addition of genetic material from a donor organism where the material is well characterized and contains only non-coding regulatory regions.” Release into the environment (Federal Register 52:22908–9) “The use of a regulated article outside the constraints of physical containment that are found in a laboratory, contained greenhouse, or a fermenter or other contained structure.” Well-characterized and contains only noncoding regulatory regions (APHIS 1987; e.g., operators, promoters, origins of replication, terminators, and ribosome binding regions) “The genetic material added to a microorganism in which the following can be documented about such genetic material: (a) The exact nucleotide base sequence of the regulatory regions and any inserted flanking nucleotides; (b) The regulatory regions and any inserted flanking nucleotides do not code for protein or peptide; and (c) The regulatory region solely controls the activity of other sequences that code for protein or peptide molecules or act as recognition sites for the initiation of nucleic acid or protein synthesis.” ring organisms, which might be plant pests. For example, APHIS regulates the movement and release of geographically separated populations of known plant pests because the source population can have characteristics that are absent in the recipient geographic population, which could

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation increase the plant pest risk in the recipient population. In all cases a permit must be obtained from APHIS before importation and interstate movement of potential plant pests. For some transgenic organisms, APHIS has determined that release into the environment is tantamount to introduction of a new organism; therefore, it requires that a permit be obtained beforehand. In other words, APHIS considers that transgenic organisms are new organisms because they potentially have new ecological characteristics that could make them plant pests. That is consistent with the principle that transgenic organisms might have new characteristics that require oversight and that the genetic engineering process might trigger oversight, even if it is not considered in the evaluation of risks, as discussed in detail in Chapter 2. In 1993 APHIS proposed a simplification of the regulatory procedure in order to: create a simplified notification procedure for transgenic plants that meet eligibility criteria and are tested using performance standards that minimize risks; allow under notification corn, cotton, potato, soybean, tobacco, tomato, or any additional plant that BBEP has determined may be safely introduced; extend notification procedures to include a number of virus resistance modifications; provide a petition process allowing for a determination that certain plants are no longer regulated articles; and allow permissions for determination for nonregulated status to be extended to closely related articles (an “extension” process). APHIS received 84 comments ranging from complete opposition to enthusiastic support for even greater reduction in regulation. According to the Federal Register notice (APHIS 1993), the majority of commenters expressed general or qualified support for the suggested changes. Prior to this proposed change, however, APHIS consulted with a scientific subcommittee of the USDA’s Agricultural Biotechnology Research Advisory Committee (ABRAC) in a public forum about the scientific basis for the proposed reduction in regulation. APHIS argued that the six specified crops had no weedy relatives, that only cotton had wild populations (in Florida), and that only cotton and potato had wild relatives in the United States. It also argued that the confinement criteria in the performance standards should protect against gene flow to these wild relatives and populations, and the subcommittee generally concurred. (ABRAC held its last meeting in January 1996.)

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation In 1995 APHIS proposed a further simplification of the regulatory procedure to: allow environmental release under the notification system of any plant not considered a noxious weed as long as the plant is not considered a weed in the intended area of release; extend the notification procedures to include a number of genetic modifications resulting in virus resistance; discontinue the requirements for each relevant state to concur with interstate movement; simplify reporting requirements for field trials conducted under notification or permit; and allow permissions for determination of nonregulated status to be extended to closely related articles (an “extension” process). These proposed changes also proved to be controversial. A total of 50 comments were received from industry, universities, state departments of agriculture, science policy organizations, environmental groups, industry organizations, professional societies, consumer organizations, individuals, and a university cooperative extension office. The majority (over 60%) supported the proposed amendments. Those expressing opposition were concerned with the potential for increased risk posed by particular transgenic plants, especially those with wild or weedy relatives. Some opponents were concerned that regulatory oversight would be decreased in the shift from a permitting to a greater emphasis on the notification system and that notification was harder to enforce and therefore compliance might be compromised. Part of the APHIS response to the expressed public concerns was publication of a revised user’s guide (APHIS 1997b), which provided examples of how applicants might meet the performance standards and other information to help applicants design their field trials for specific organisms. Although not a formal regulatory document, the user’s guide provides information to applicants on how to comply with the regulations. The guide gives specific examples and describes approaches to compliance on such matters as shipping and maintenance of regulated articles, how to avoid admixtures, devitalizing treatments, elimination of viable vectors, and minimizing dispersal and persistence in the environment. The guide emphasizes that these are examples only, that following the examples exactly does not necessarily ensure acceptability, and that alternative approaches might be equally acceptable. APHIS also argued that field inspections of trials conducted under the notification system (as well as those under permit) achieved a high degree of compliance.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation The controversial changes proposed in 1995 that went into effect in 1997 were created by APHIS internally, and the scientific response of APHIS also was internal. This subcommittee recognizes that the proposed changes of 1995 were precedent setting (e.g., extending the notification procedure from six crops to all nonweed plants) and created without external scientific input beyond those respondents to the Federal Register notice. It is certainly possible that APHIS may continue to propose and make important policy changes without external scientific input. Recommendation 3.1: For changes in regulatory policy, APHIS should convene an external scientific advisory group and hold at least one meeting to solicit public scientific input to review proposed changes. SCOPE AND REGULATORY PROCEDURES USED BY APHIS APHIS currently regulates transgenic plants under 7 CFR part 340, “Genetically Engineered Organisms and Products: Simplification of Requirements and Procedures for Genetically Engineered Organisms,” which was published in 1997. Anyone introducing (importing, transporting interstate, or releasing into the environment) a regulated article must have authorization through either a notification or a permit and must comply with other restrictions as described in 7 CFR part 340. The regulations provide APHIS with the authority to regulate such introductions for certain transgenic organisms. The regulatory objective of 7 CFR 340 is to allow the evaluation of transgenic organisms with sufficient scrutiny to identify any plant pest risks at an early enough stage to allow remedial action prior to the occurrence of any real damage. The basis for regulation is the broadly defined plant pest. Part 340.1 provides definitions for the terms used (see BOX 3.1). Plant pest is defined as “any living stage (including active and dormant forms) 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 agents or substances, which can directly or indirectly injure or cause damage in or to any plants or parts thereof, or any processed, manufactured or other products of plants.” Under this definition, almost all organisms (and their derivatives) can be potential plant pests, including, for example, herbivorous invertebrate animals (anything that eats a plant can be considered a plant pest). A transgenic organism is considered a regulated article if it is a plant pest or if it or a gene donor or vector used in its construction are plant pests according to a long list of taxa listed in 7 CFR

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation 340.2 (this list can be amended as outlined in part 340.5). In addition, a transgenic organism can be considered a regulated article if APHIS has reason to believe it presents a plant pest risk. The definition of plant pest is in many ways extremely broad but in other ways surprisingly restricted. The breadth of the definition is provided by the inclusion of indirect injury or disease. Indirect injury occurs when a plant pest has an effect on another species that eventually leads to a detrimental effect on a plant. Thus, any species that interacts ecologically with a species that directly injures a plant (e.g., by feeding on it) can be considered a potential plant pest. Indeed, species that interact with species that interact with direct plant pests can be considered indirect plant pests, and this leads to the potential inclusion of nearly every species. In contrast, the taxonomic list restricts the definition of plant pest in an important way. According to the definitions, no vertebrate can be considered a plant pest, despite considerable evidence to the contrary. (This situation might change under the new Plant Protection Act of 2000; see Chapter 7.) For example, feral pigs and goats have had serious effects on native plants in Hawaii and other oceanic islands (Allen 2000). The exclusion also means that genetically engineered fish cannot be regulated as potential plant pests. There is some uncertainty about the status of algae as plants that could be affected by potential plant pests. Modern classification excludes most algae from the Kingdom Plantae (Campbell et al. 1997). Thus, many algae are not members of Plantae, as required by the definition of plant, but they are listed as a group under the definition. That is an important distinction because shellfish, which consume unicellular algae and are invertebrates, could be regulated under the APHIS rules if unicellular algae were considered members of the Kingdom Plantae. Note that if a transgenic plant was created from a nonweedy species without the insertion of genes from a plant pest and it was transformed without the intervention of a plant pest (as would be the case with the use of particle bombardment or electroporation), it would not necessarily be considered a regulated article. In such cases, the creators of transgenic plants to be field released apparently have always sent a “courtesy” notification or permit application to APHIS, but the possibility remains that field release of certain transgenic plants could escape APHIS oversight. Notification System for Introduction of Certain Regulated Articles (7 CFR 340.3) Most transgenic plants are field tested under a notification system. In recent years “nearly 99% of all field tests, importations, and interstate movements of engineered plants [have been] performed under this sys-

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation tem” (OSTP/CEQ 2001). Notification for the movement, importation, and field testing of transgenic plants follows a rigid but streamlined format that allows an expeditious review. For a specified list of plants and characteristics, an applicant may simply notify APHIS of its intent to release a regulated article. Upon receipt of such a notification application, the document is logged into the APHIS database and reviewed by one of the scientific staff for qualification and completeness, and then a recommendation is sent to appropriate state officials for concurrence. The entire process must be completed within 30 days (10 days for interstate movement). Scientific evaluation of a notification application by APHIS personnel is typically completed within a few days. Acknowledgment by APHIS that an article meets the notification requirements means that a permit is not required for field testing within one year of the date of introduction. Renewal can be accomplished by submitting an additional notification to APHIS. The assessment of notifications is not subject to external scientific review or any other public input. A few transgenic plants are now grown to produce commercial products under notification. To meet APHIS criteria for notification: The plant must not be listed as a noxious weed under the Federal Noxious Weed Act or be considered to be a weed in the area of release into the environment. The inserted DNA must be stably integrated into the host genome. According to the user’s guide (APHIS 1997bs), this means that the trait is inherited in a Mendelian fashion for at least two generations. The function of the inserted DNA is known, and its expression does not result in plant disease. Function is not precisely defined in the user’s guide, but the intent of this criterion is that expression does not result in plant disease. The inserted DNA does not cause production of an infectious entity; encode substances that are known or likely to be toxic to nontarget species known or likely to feed on the plant (According to the user’s guide, toxicity to non-target species is restricted only to those nontarget species that feed on the plant, not dispersed plant parts, such as seeds, pollen, or plant residue.); encode products intended for pharmaceutical use. (According to the user’s guide, “intention” becomes clear for regulatory purposes only when clinical testing of the product is proposed to the Food and Drug Administration. Until such time, a product is not considered to be intended for pharmaceutical use.)

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation Any virus-derived sequences must be known noncoding regulatory sequences or otherwise unlikely to facilitate virus virulence and spread in plants. The inserted sequence must not be derived from human or animal viral pathogens or other potential human or animal disease-causing agents. In addition to meeting these criteria, introductions under the notification system must meet specified performance standards designed to ensure such confinement that the transgenic plant or its progeny will not unintentionally persist in the environment. The standards state general concerns but leave applicants the flexibility to meet them according to their own circumstances: The transgenic plant must be transported and stored in a way that minimizes the escape of viable plant parts into the environment. According to the APHIS user’s guide, this performance standard refers to seeds, cuttings, buds, and other plant parts that would be planted to grow the crop but not the movement of pollen. For environmental release, inadvertent mixing of the transgenic plant with nonregulated plant material must be avoided. According to the APHIS user’s guide, this performance standard refers to seeds, cuttings, buds, and other plant parts that would be planted to grow the crop but not the movement of pollen. The identity of the transgenic plants and their parts must be maintained; plants and their parts must be contained or devitalized after use. According to the APHIS user’s guide, this performance standard does not cover the movement of pollen. There must be no pathogenic vector associated with the plant. Field trials must be conducted in a manner that precludes persistence of the plant and its progeny. Management practices to prevent persistence of the plant or its progeny in the environment must be applied. APHIS provides detailed guidance for notification applications in its user’s guide including sample notification letters and guidance on how to meet performance standards. The notification application consists of the name and identity of the responsible person (applicant) and a description of the regulated article. The description must include the identity of the transformed plant species, the method of gene transfer, and a full description of the inserted sequences, including the functions, encoded proteins, and source donor organisms for each segment. In addition, the notification application must include, for field releases, the geographic details, size, and duration of field trials and, if appropriate, information about

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation intended transport. Finally, the applicant must sign a statement affirming that the article meets the eligibility requirements and that any actions taken will meet the mandated performance standards listed above. Any applicants whose notification application is denied may apply, without prejudice, for a permit for the same regulated article. Permits for Introduction of a Regulated Article (CFR 340.4) Permits are required for the movement, importation, and field testing of transgenic plants that do not qualify for notification (such as pharmaceutical-producing plants) and for plants denied notification. Under the permitting process described in CFR 340.4, APHIS presumes that the regulated article is a potential plant pest and requires anyone who wants to introduce it into the environment to obtain a permit. The permitting process allows APHIS to evaluate the potential plant pest risk and prescribe prevention measures to reduce it (e.g., through confinement procedures, or limiting the spatial, temporal, and numerical scales of the release). At least one regulated article is now grown to produce a commercial product under permit. It is anticipated that commercial production of pharmaceutical products will occur under the permitting process. The process provides APHIS with the authority to request further information from applicants, but it does not give the agency the authority to require the requested information. Although applicants are not legally required to provide additional requested information, so far all have complied, presumably to maintain their desired cooperative relationship with APHIS. The application for a permit for release into the environment is more detailed than the notification application. The primary emphasis for field release under permit is information regarding confinement. That is, the confinement imposed should effectively eliminate the potential for significant environmental impact. An application for a permit must provide data so that a decision can be made to ensure that (a) the transgenic plant is adequately characterized; (b) no transgenic plant material will persist in the environment; (c) that unintentional or unanticipated effects, if any, can be restricted to the receiving facility or the confined field site; and (d) in the case of field testing, plants must be managed in such a way that there are no environmental risks after the confined field release is terminated. APHIS must receive field trial results within six months of trial completion. The applicant must allow APHIS and state inspectors access to the trial and must notify the agency of any unusual occurrences. APHIS provides detailed guidance for permit applications in its user’s guide including sample applications. In the permit application the applicant lists the regulated article; the donor organism; vector or vector agent;

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation date of importation, movement, or release; and port of importation or site of release. Additionally, detailed information is required, as appropriate, on the: anticipated or actual expression of the altered genetic material in the regulated article and how it differs from the nonmodified parent organism; molecular biology of the system; locality where the donor, recipient, and vector were collected and produced; experimental design at the release site; facilities at the destination; measures to ensure confinement; and final disposition of the regulated article. The permit application should be submitted to APHIS at least 120 days prior to intended release into the environment. APHIS conducts an initial review of the dossier (within 30 days) to determine if all necessary information is supplied. If not, the 120-day “clock” stops until the applicant provides the missing information. The permitting process allows APHIS to request monitoring and reporting of the results of small-scale releases. Petition for Determination of Nonregulated Status (CFR 340.6) APHIS has a procedure whereby an applicant can request that the agency determine that a particular transgenic plant is not a regulated article—that is, that it does not fall under the definition of a plant pest (BOX 3.1). This procedure is the sole route for commercialization of transgenic plants (e.g., sale of transgenic soybean seed) and the primary but not sole route to commercialization of transgenic plant products (e.g., when the plants are never sold but a product such as an industrial protein extracted from the plant is sold). Once APHIS decides that a transgenic plant is not a regulated article, it cannot exercise any additional oversight on the plant or its descendants. Those descendants include all plants of the same species that receive the transgene through sexual reproduction. Therefore, separate deregulated lines can be mated with one another via conventional crossbreeding to bring together different transgenes in the same plant, and such plants are not subject to regulatory evaluation (see discussion of methods to create multitransgenic plants in Chapter 4). Descendants may also include distantly related members of the same crop species. For example, after deregulation, a breeder could use cross-

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation ing to move a transgene not only from one flax variety to another flax variety, or from one cabbage to another, but also from flax to linseed and from cabbage to broccoli. Although it is difficult to move genes among more distant relatives through crossing, it is not impossible. For example, crosses among different species have been used to create modern cultivars of sugarcane and raspberry (Smartt and Simmonds 1995; see also Chapter 1). Finally, intended or unintended crosses may result in descendants that are wild species, when those species receive the transgenes from introgression from the crop. Also, APHIS cannot require monitoring of any deregulated article. Finding 3.1: Currently, APHIS deregulation is absolute, completely removing the article from the agency’s regulatory authority. Finding 3.2: Presently, APHIS deregulation includes all progeny and descendants of a deregulated item. Decisions to deregulate articles are evaluated on a case-by-case basis; the procedural requirements are specified in the Federal Register (APHIS 1993). APHIS has made decisions to deregulate dozens of genetically modified crops. The current list of deregulated crops (and notification and permit applications) can be obtained at a website maintained by Information Systems for Biotechnology (www.nbiap.vt.edu/). Anyone can petition, with supporting evidence, that a specified transgenic plant be determined a nonregulated article. Essentially, the applicant must show that the regulated article is free from any risk under 7 CFR 340. Petitions for determination of nonregulated status are comprehensive data packages to APHIS for scientific review. Assessment of petitions by APHIS scientists relies on data supplied primarily or exclusively by the applicant, but APHIS may seek additional information (the case study on squash in Chapter 4 is an example). The accumulated information is used to determine whether the regulated article displays no plant pathogenic characteristics; is not more likely to become a weed than its nontransformed parent; is unlikely to increase the weediness of cultivated, feral, or wild-related plants; does not damage processed agricultural commodities; and is unlikely to cause unintended significant harm in other organisms. Because reviews are conducted on a case-by-case basis, the information needed may vary with plant species, the specific type of modification(s), and end use of the transgenic plant. The following is a summary of the required data and information in the Federal Register (APHIS 1987). a description of the biology and taxonomic identification of the nonmodified recipient plant;

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation relevant experimental data and publications; a detailed description of the genotypic differences between the regulated article and the nonmodified recipient organism; a detailed description of the regulated article’s phenotype, especially regarding known and potential differences from the nonmodified recipient organism that would indicate whether or not the regulated article poses a greater plant pest risk; and field test reports for trials involving the regulated article conducted under permit or notification. To provide guidance for petitions, the APHIS user’s guide has a sample application for determination of nonregulatory status representing a suggested format for submission. APHIS has inserted in the left-hand margin of the sample application comments and issues that may need to be addressed by the applicant. The guidance detailed under each heading is directly quoted below from the user’s guide: I. Rationale for Development of the Product II. Relevant Biology of the Plant Description of the biology of the nonmodified recipient organism should include taxonomy, genetics, pollination, evidence of reported weediness (e.g., noting whether the crop or sexually compatible species is listed in the relevant publications of the Weed Society of America), discussion of sexual compatibility with wild and weedy free-living relatives in natural crosses or crosses with human intervention. The applicant should provide source of recipient (cultivar name or accession number) and the weed status of its sexually compatible relatives. The applicant should explicitly identify the lines that are to be considered in the petition and the cultivars from which they are derived. If there are multiple lines, each line must be given a unique identifier that must be listed in the application. For virus-resistant plants, applicants should provide in an additional section the following information on the nature of the virus that provided the sequences encoding the resistance phenotype: the taxonomic name of the virus including family, genus, and strain designation including any synonyms; the type of nucleic acid contained in the virus; whether the infection is systemic or tissue specific; whether the virus is associated with any satellite or helper viruses;

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation the natural host range of the virus; how the virus is transmitted; if transmitted by a vector, the identity of the vector including mode of transmission (e.g., persistent or non-persistent); whether any synergistic or transcapsidation interactions with other viruses under field situations have been reported in the literature; and the location and the name of the host plant the virus was originally isolated from. The above information can be provided in a table format. This information can be supplemented by listing references that report the host range, insect vectors, etc., for the virus. III. Description of the Transformation System For Agrobacterium-based transformation protocol, the applicant must indicate how Ti plasmid-based vector was disarmed (i.e., all tumorigenic DNA was removed). Applicants can provide citations that describe the transformation procedure. However, any significant modifications of transformation, strain designation, etc., should be described. For other methods of transformation, the applicant can describe the sources of various components of the plasmid (or other DNA including possible carrier DNA) and method of transformation by citation. However, any significant modifications of transformation, strain designation, etc., should be described. The applicant must provide a detailed restriction map of the plasmid that is sufficient to be used in the analysis of Southern data. Description of added restriction sites is helpful in the interpretation of Southern data and should be provided. Indicate the functions of the gene(s), promoters, leader sequences, enhancers, introns, and any other sequences that are used for gene expression in the plant and a reference describing from where the sequences were obtained. Discussion should include whether the inserted sequences are responsible for disease or injury to plants or other organisms. The nucleotide sequence(s) of the plant-expressed gene(s) should be provided by citation and not submitted in the application. If there has been a significant modification to sequences and the modified sequence has not been published, they should provide the complete sequence highlighting the modifications. If there have been minor modifications to the sequence of the plant-expressed gene, they should be provided. For example, if in a chemically synthesized Bt gene amino acid 23 was changed from methionine to alanine, it should be stated without providing the complete sequence.

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation IV. Donor Genes and Regulatory Sequences V. Genetic Analysis and Agronomic Performance In general, it always is prudent to analyze data statistically when such analysis is possible. When unpublished information or an opinion has been supplied by a scientific expert, a letter communicating the information should be included in the petition. If the unpublished information provided is data resulting from scientific research then these data can be provided as a personal communication either in a letter from the researcher or in the text of the petition. In either case the materials and methods, data analysis, and discussion of the data analysis should be provided in detail. Unsupported assertions about the results of the experiment are not acceptable. Applicants must report any differences noted between transgenic and nontransgenic plants that are not directly attributed to the expected phenotype. Differences observed could include changes in leaf morphology, pollen viability, seed germination rates, changes in overwintering capabilities, insect susceptibilities, disease resistance, yield, agronomic performance, etc. Applicants must also note the types of characteristics that were compared between transgenic and nontransgenic plants and found to be unchanged. The applicant should describe whether data submitted are from inbred or hybrid plants; if hybrid plants, state which generation. The applicant should state whether data with respect to plant performance were generated in a greenhouse or field environment, and if from the field, indicate how many sites, states and number of years the data represents. Seed germination, seed dormancy, seed production, growth rate, and other data relating to the plant’s performance will be required when the nature of the gene and the biology of the plant (including sexually-compatible relatives) warrant such data. This type of data will usually not be required for plants that have some of the following attributes: highly domesticated (e.g., corn), exclusively self-pollinating (e.g., soybean), male sterile, and have high seed germination rates (>90%), and whose phenotypes are unlikely to affect performance with respect to weediness or fitness (e.g., delayed ripening or oil seed modification). Phenotypes that might require performance data (depending on the plant) include but are not limited to the following: cold tolerance, salt tolerance and tolerance or resistance to other biotic or abiotic stresses. Southern analysis should include DNA isolated from nonmodified recipient, all or selected transformed lines, and the vector. Parental plasmid DNA (e.g., PUC 18) not containing intended donor genes may be

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation labeled and hybridized to Southern blots to demonstrate that only the intended sequences have been incorporated in the genome of the transgenic plant. Restriction enzymes to be used might include enzymes that do not cut within the transforming plasmid but will cut the “entire insert” into one fragment from the DNA of the transgenic plant. In the case of an Agrobacterium-based transformation system, the applicant should determine if genes that reside outside the LB/RB are inserted in the genome of the regulated cultivar. If a complete copy of any of these genes is present, the applicant should determine whether it is expressed in the plant. For direct transformation systems, applicants should determine which sequences are inserted in transgenic plants and whether they are expressed. PCR analysis may be used to prove that only the targeted DNA has been incorporated. Sequencing of the transgene in plant and adjacent sequences is not required. Determination of the number of copies of integrated transgenes is not required, but the number of insertions may be used to support analysis of inheritance data. If the inserted DNA sequence order is complex, as is often the case for plants engineered via direct transformation systems (e.g., electroporation, polyethylene glycol transformation of protoplasts, or particle bombardment techniques), the applicants should summarize the data by providing the following information for all the genes (whether under the direction of plant or bacterial promoters). Is there a complete copy of the gene present in the regulated article? Is the protein expressed in the plant? If multiple complete copies of a gene are present, applicants do not have to determine if each copy of the gene is expressed. Applicants should provide a table, like the one shown below [the user’s guide provides a table with hypothetical data], that summarizes the results and indicates where specific data is to be found. Mendelian inheritance data and Chi square analysis for at least 2 generations are appropriate to demonstrate whether the transgene is stably inserted and inherited in Mendelian fashion. Such data are generally not necessary for infertile vegetatively propagated crops such as male-sterile potatoes. RNA—Northern analysis is generally not required except for virus-resistant plants. However, such analysis may be necessary for ribozyme, truncated sense, or antisense constructs, when protein levels cannot be provided. PROTEINS—Expression levels of gene(s) of interest and marker genes in various tissues, developmental stages of plant, and experimental conditions (induced or noninduced) are required. Assays can be of enzyme activity. Serology, ELISA, and Western blots may also be used. Describing the source of the immunogen is critical for serological analysis. For virus resistant plants, the amount of viral transgene RNA produced should be determined and compared to the amount of the RNA

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation produced by the viral gene in an infected nontransgenic plant. Applicants should address whether the transgene RNA (or protein) is present in the same tissues as are infected during natural infections. From transgenic plants singly infected with each of the widely prevalent viruses in the U.S. that normally infect the recipient plant (contact APHIS for the list of these viruses) determine the amount of both coat proteins (i.e., from both the transgene and the naturally infecting virus). For comparison, provide the amount of both coat proteins produced in the nonengineered plant in mixed infections of the virus from which the coat protein gene was derived and the same widely prevalent viruses used in the single infection study. Provide description of symptoms of infected plants in all cases. For all diseases and pathogens surveyed, names of the diseases and the scientific names of the pathogens should be provided. Data from field tests in foreign countries are acceptable. If the data on diseases and pests were obtained in the foreign country, the applicant should submit information about the distribution of those pests, disease or pathogens in the U.S. Disease and pathogen susceptibility on wild type and transgenic plants should be determined preferably from natural infestations. However, if the applicant must use direct inoculations, i.e., with virus resistant transgenic plants, the source and taxonomic classification of the virus should be provided. Certain plants have minute quantities of known toxicants which may adversely impact nontarget organisms and beneficial insects; e.g., tomatine in tomatoes, cucurbitin in cucurbits (APHIS identified cucurbitin as a known toxicant while they probably meant curcurbitacin. Cucurbitin is an amino acid that to the subcommittee’s knowledge is not highly likely to affect nontarget species, whereas cucurbitacins are highly toxic “bitter principles” long known to have effects against both herbivores and predators (which may attack cucurbitacin-sequestering herbivores), gossypol in cotton, etc. If such plants are recipients of transgenes, the applicant should provide information as to whether the level of toxicants is altered. If the plant produces no known toxicant, the applicant should state so and provide the reference to support the claim. Plant toxins can be assessed by the tests and criteria that plant breeders traditionally use in the crop. In some instances, this may be done qualitatively, e.g., taste testing of cucurbits. VI. Environmental Consequences of Introduction of the Transformed Cultivar VII. Adverse Consequences of Introduction Assuming that the levels of known toxicants in the regulated article reported in Section V are in acceptable range; that there were no notable

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation differences reported in Section V between transgenic and nontransgenic plant; and that the gene(s) engineered into the recipient plant have no known reported toxic properties; then, toxicological data on effects of the plant on nontarget organisms and threatened and endangered species will usually not be required. A separate petition should be submitted for each category/phenotype combination. For example, a petition for coleopteran insect resistant potatoes or PVY resistant potatoes should be submitted separately. However, when a single plant contains more than one phenotype modification, submit only one petition. For example, one petition should be submitted for potatoes that are both PVY and PVX resistant. Upon receiving a petition for nonregulated status, APHIS assigns a petition number and publishes a notice in the Federal Register to solicit comments from the public. Comments submitted within the 60-day comment period become part of the file. Within a 180-day period, the administrator responds in writing to the petitioner, either approving (in whole or in part) the petition or denying it. If the administrator denies a petition, the reasons are provided in writing to the applicant. The unsuccessful applicant may appeal the decision within 10 days of receipt of the written notification of denial. Requests for Extension of Determination of Nonregulated Status to Additional Regulated Articles APHIS may extend a determination of nonregulated status to additional regulated articles if it finds that such articles do not pose a potential for plant pest risk. Extension requests are handled somewhat like petitions. Under the extension provisions, the administrator may determine that a regulated article is substantially similar to another nonregulated article and does not pose a plant pest risk. Any person may request consideration under this argument to other regulated articles, based on submitted evidence of similarity to equivalent nonregulated articles. The finding is based on an evaluation of the similarity of the additional regulated articles to an organism that has already been the subject of a determination of nonregulated status by APHIS and is used as a reference (antecedent) for comparison to the regulated article under consideration. In its user’s guide, APHIS provides examples that illustrate molecular manipulations that create organisms the agency believes are unlikely to pose new risks beyond those that would have already been considered in the initial determination of nonregulated status: “Modifications in which the amino acid sequence of any encoded proteins is unchanged with respect to the corresponding sequence in the antecedent organism (i.e., synonymous codon changes).”

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation “Production of new transformants of the same cultivar as the antecedent organism, or new cultivars of the same plant species or variety that do not differ significantly from the antecedent cultivar in reproductive fitness, obtained via transformation using the same transforming nucleic acid as was used in producing the antecedent organism.” “Production of new transformants of the same cultivar as the antecedent organism, using a transformation vector that differs from that used to produce the antecedent organism only: in noncoding regulatory sequences used to control expression of any of the introduced genes; or in other vector DNA sequences that were not incorporated into the recipient plant cells, unless the new regulatory sequences cause the expression of any introduced genes in plant tissues in which the introduced genes were not expressed in the antecedent organism.” “Production of new transformants of the same cultivar as the antecdent organism, in which the genetic material transferred into the recipient plant to produce the antecedent organism is identical to that in the regulated article in question, but in which said material was introduced into the recipient plant using a different transformation vector or technique.” “Modifications in which the antecedent organism and the regulated article in question contain different donor genes, but the donor gene used in producing the antecedent organism and the donor gene used in producing the regulated article in question encode enzymes catalyzing the same biochemical reaction (i.e., molecules that have the same substrates and products) or encode other proteins performing the same molecular function (i.e., molecules that bind to the same target molecule in vitro and either inhibit its function via the same mechanism, or cause the same biochemical change in the target molecule).” “Modifications in which the antecedent organism and the regulated article in question differ only in [the] marker genes they contain, which were used in their identification or selection, provided that the new marker genes in the organism in question do not raise any new risk issues, i.e., do not encode substances toxic to plants; do not encode substances with pesticidal properties; do not confer resistance to any antibiotic of significant importance for veterinary or human use; and do not confer resistance to any different herbicide than was conferred by the marker genes in the antecedent organism.” To provide guidance for extension requests, the APHIS user’s guide provides a sample request, which suggests that certain information be provided by the applicant. Essentially, the request is a streamlined petition that focuses on differences between the regulated article and the antecedent organism. In particular, APHIS focuses first on a “precise description

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Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation of the genetic modifications in the regulated articles under consideration and detailed comparison of the modifications in those regulated articles with those in the antecedent organism.” For organisms not covered in the APHIS examples above, the applicant may address why the particular organisms in question do not raise any issues different from those considered in the determination for the antecedent organism, thereby meriting separate consideration under a new petition. Also, the requesters should provide information on the phenotypic expression of the genetic modifications in the regulated articles, indicating any expected or unexpected differences in phenotype between the regulated articles and their antecedent organism. Data from at least one field trial should be included, and all data reports from completed field trials with a regulated article should be submitted with the extension request. CONCLUSION APHIS regulation of transgenic organisms has evolved over the past two decades. To meet the growing number of field tests an expeditious notification system has evolved, assuring rapid turnover of field test applications. The permitting process, originally used for all field tests, is now used to deal with field testing situations that require substantial scrutiny (such as products intended for pharmaceutical use). Both the notification and permit processes require that transgenic organisms be grown and handled in such a way as to prevent their escape into the ambient environment. Petitions of the determination of nonregulated status (and a similar extension system) represent what has been the primary pathway to commercial plantation of transgenic plants. However, commercial products have also been created from regulated transgenic organisms that have been grown under notification and permit. It is not clear which of these regulatory pathways will become the option of choice for future field-based commercial plantations of transgenic plants.