Response to Monsanto Company Petition for
Determination of Nonregulated Status for Insect-
protected Corn line MON 80100
Prepared by
United States Department of Agriculture
Animal and Plant Health Inspection Service
Biotechnology, Biologics, and Environmental Protection
TABLE OF CONTENTS
I. SUMMARY1
II. BACKGROUND2
A.APHIS regulatory authority.2
B.EPA and FDA regulatory authority.4
III. RESPONSE TO COMMENTS4
IV. ANALYSIS OF THE PROPERTIES OF CORN LINE MON 801004
A.The introduced genes, their products, and the added regulatory sequences do not present a plant pest risk in corn line MON 80100.7
B.Expression of the insect control protein in corn line MON 80100 will not likely provide a competitive advantage sufficient to cause these plants to become any more "weedy" than other corn9
C.Gene introgression from corn line MON 80100 into wild or cultivated sexually compatible plants is unlikely, and such rare events should not increase the weediness potential of resulting progeny or adversely impact biodiversity.10
D.Use of corn line MON 80100 should have no more adverse impacts on raw or processed agricultural commodities than the parent corn11
E.Corn line MON 80100 exhibits no significant potential to harm organisms beneficial to agriculture, to harm threatened or endangered organisms or to have an adverse impact on the ability to control nontarget insect pests..11
F.Cultivation of Corn line MON 80100 should not reduce the ability to control insects in corn and other crops15
V. CONCLUSION17
VI. LITERATURE CITED19
I.SUMMARY
APHIS regulations promulgated pursuant to authority granted by the Federal Plant Pest Act (FPPA), and the Plant Quarantine Act (PQA), govern the introduction (importation, interstate movement, or release into the environment) of certain genetically engineered organisms and products. An organism is no longer subject to these regulatory requirements when it is demonstrated not to present a plant pest risk. The regulations provide that a person may petition the agency to evaluate submitted data and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated.
Based on a review of scientific data and literature, the Animal and Plant Health Inspection Service (APHIS) has determined that lepidopteran insect-protected corn (hereafter referred to as corn line MON 80100) does not present a plant pest risk and is therefore no longer a regulated article. APHIS made this determination in response to a petition received on April 3, 1995 from Monsanto Company. The petition requested a determination from APHIS that corn line MON 80100 does not pose a plant pest risk and therefore, is not a regulated article. APHIS announced receipt of the petition in the Federal Register (60 FR 30061-30062) on June 7, 1995 and stated that the petition was available for public view. APHIS invited written comments on this proposed action, to be submitted on or before August 7, 1995. As a result of this determination, oversight under 7 CFR Part 340 will no longer be required by APHIS for field testing, importation, or interstate movement of corn line MON 80100 or its progeny.
Corn line MON 80100 is genetically engineered to carry a gene that codes for an insecticidal protein naturally produced by the soil bacterium Bacillus thuringiensis subsp. kurstaki (Btk), together with a selectable marker genes encoding phosphinothricin acetyltransferase from Streptomyces hygroscopicus. These genes also have accompanying DNA regulatory sequences that modulate their expression. The regulatory sequences were derived from corn and the plant pathogenic cauliflower mosaic virus (CaMV) and Agrobacterium tumefaciens.
This determination has been made based on an analysis that revealed that corn line MON 80100 plants: 1) exhibit no plant pathogenic properties, 2) are no more likely to become a weed than insect-resistant corn developed by traditional breeding, 3) are unlikely to increase the weediness potential of any other cultivated plant, 4) do not cause damage to processed agricultural commodities, 5) are unlikely to harm other organisms that are beneficial to agriculture or to adversely impact the ability to control nontarget insect pests, and 6) are unlikely to reduce the ability to control insects in corn and other crops. APHIS has also concluded that there is no reason to believe that new corn varieties derived from corn line MON 80100 progeny will exhibit new plant pest properties; i.e., properties substantially different from any observed for the corn line MON 80100 already field tested, or those observed for corn in traditional breeding programs.
The potential environmental impacts associated with this determination have been examined in accordance with regulations and guidelines implementing the National Environmental Policy Act (NEPA) of 1969 (42 U.S.C. 4331 et seq.; 40 CFR 1500-1509; 7 CFR Part 1b; 44 FR 50381-50384; and 44 FR 51272-51274). An environmental assessment (EA) was prepared and a preliminary Finding of No Significant Impact (FONSI) was reached by APHIS for the determination that corn line MON 80100 is no longer a regulated article under its regulations at 7 CFR Part 340. This decision does not release corn line MON 80100 from regulations administered by the Environmental Protection Agency (EPA) under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136 et seq.) and the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 301 et seq.).
The body of this document consists of three parts: (1) background information that provides the legal framework under which APHIS has regulated the field testing, interstate movement, and importation of insect-protected corn; (2) a summary of, and response to, comments provided to APHIS on its proposed action during the public comment period; and (3) analysis of the key factors relevant to APHIS' decision that insect-protected corn does not present a plant pest risk.
II.BACKGROUND
A. APHIS Regulatory Authority
APHIS regulations at 7 CFR 340, which were promulgated pursuant to authority granted by the Federal Plant Pest Act (FPPA), (7 U.S.C. 150aa-150jj) as amended, and the Plant Quarantine Act (PQA), (7 U.S.C. 151-164a, 166-167) as amended, regulate the introduction (importation, interstate movement, or release into the environment) of certain genetically engineered organisms and products. Under this regulation, a genetically engineered organism is deemed a regulated article either if the donor organism, recipient organism, vector or vector agent used to develop the organism belongs to one of the taxa listed in the regulation and is also a plant pest; or if APHIS has reason to believe that the genetically engineered organism presents a plant pest risk. The FPPA gives U.S. Department of Agriculture (USDA) the authority to regulate plant pests and other articles to prevent direct or indirect injury, disease, or damage to plants and plant products. In addition, the PQA provides an additional level of protection by enabling USDA to regulate the importation and movement of nursery stock and other plants that may harbor injurious pests or diseases.
Before the introduction of a regulated article, a person is required under Section 340.0 of the regulations either to (1) notify APHIS in accordance with Section 340.3 or (2) obtain a permit in accordance with Section 340.4. Introduction under notification (Section 340.3) requires that the introduction meet specified eligibility criteria and performance standards. The eligibility criteria impose limitations on the types of genetic modifications that qualify for notification. The performance standards impose limitations on how the introduction may be conducted. Under Section 340.4, a permit is granted for a field trial when APHIS has determined that the conduct of the field trial, under the conditions specified by the applicant or stipulated by APHIS, does not pose a plant pest risk.
An organism is not subject to the regulatory requirements of 7 CFR Part 340 when it is demonstrated not to present a plant pest risk. Section 340.6 of the regulations, entitled "Petition Process for Determination of Nonregulated Status," provides that a person may petition the agency to evaluate submitted data and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If the agency determines that the regulated article does not present a risk of introduction or dissemination of a plant pest, the petition will be granted, thereby allowing for unregulated introduction of the article in question. A petition may be granted in whole or in part.
The Corn line MON 80100 has been considered a "regulated article" for field testing under Part 340 of the regulations in part because certain noncoding regulatory sequences were derived from CaMV and Agrobacterium tumefaciens, known plant pests. APHIS believes it prudent to provide assurance before commercialization that organisms such as corn line MON 80100, which are derived at least in part from plant pests, do not pose any potential plant pest risk. Such assurance may aid the entry of new plant varieties into commerce or into breeding and development programs. The decision by APHIS that Corn line MON 80100 is not a regulated article is based in part on evidence provided by Monsanto Company concerning the biological properties of corn line MON 80100 and its similarity to other varieties of corn grown using standard agricultural practices for commercial sale or private use.
The fact that APHIS regulates genetically engineered organisms having plant pest components does not carry with it the presumption that the presence of part of a plant pest makes a whole plant a pest or that the plants or genes are pathogenic (McCammon and Medley, 1990). APHIS' approach to plant pest risk is considerably broader than a narrow definition that encompasses only plant pathogens. Other traits, such as increased weediness, and harmful effects on beneficial organisms, such as earthworms and bees, are clearly captured by the intended meaning of direct or indirect plant pest risk. In APHIS' regulations at 7 CFR Part 340, a "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 disease or damage in or to any plants or parts thereof, or any processed, manufactured, or other products of plants."
A determination that such insect-protected organisms do not present a plant pest risk can be made under this definition, especially when there is evidence that the plant under consideration: 1) exhibits no plant pathogenic properties; 2) is no more likely to become a weed than insect-protected corn developed by traditional breeding; 3) is unlikely to increase the weediness potential of any other cultivated plant; 4) does not cause damage to processed agricultural commodities; 5) is unlikely to harm other organisms that are beneficial to agriculture or to adversely impact the ability to control nontarget insect pests; and 6) is unlikely to reduce the ability to control insects in corn and other crops. Evidence has been presented by Monsanto Company that bears on these topics. In addition, it should be established that there is no reason to believe that any new corn varieties bred from corn line MON 80100 will exhibit plant pest properties substantially different from any observed for corn in traditional breeding programs, or as seen in the development of corn line MON 80100 already field tested. APHIS does anticipate that there will be new corn varieties bred from corn line MON 80100.
B. EPA and FDA regulatory authority
The corn line MON 80100 lines are currently subject to regulations administered by the EPA and/or the FDA (described in Section II.C. of the Environmental Assessment) that require registration of pesticides prior to their distribution and sale and establish tolerances for pesticide residues in raw agricultural products. APHIS' decision on the regulatory status of the corn line MON 80100 under APHIS' regulations at 7 CFR 340, in no way releases this corn and its progeny from EPA and FDA regulatory oversight.
III.RESPONSE TO COMMENTS
APHIS received nine comments on the Monsanto petition from farmers, industry, and a university research center. All the commenters expressed support for the subject petition for nonregulated status.
IV.ANALYSIS OF THE PROPERTIES OF CORN LINE MON 80100
A brief discussion of corn biology follows in the next paragraph to help inform the subsequent analysis. This information is expanded in subsequent sections when it is relevant in addressing particular risk assessment issues.
Zea mays Linnaeus, known as maize throughout most of the world, and as corn in the United States, is a large, annual, monoecious grass, that is grown for animal feed, silage, vegetable oil, sugar syrups, and other uses. Corn is grown commercially in almost all States of the United States (Jewell, 1989). Corn has been cultivated since the earliest historic times from Peru to central North America. The region of origin is now presumed to be Mexico (Gould, 1968). Dispersal to the Old World is generally deemed to have occurred in the sixteenth and seventeenth centuries (Cobley and Steele, 1976); however, recent evidence indicates that dispersal to India may have occurred prior to the twelfth and thirteenth centuries by unknown means (Johannessen and Parker, 1989).
Zea is a genus of the family Gramineae (Poaceae), commonly known as the grass family. The genus consists of four species: Z. mays, cultivated corn and teosinte; Z. diploperennis Iltis et al., diploperennial teosinte; Z. luxurians (Durieu et Asch.) Bird; and Z. perennis (Hitchc.) Reeves et Mangelsd., perennial teosinte. Variations of the species have been assigned to the segregate genus Euchlaena, which is not currently recognized, or have been divided into numerous small species within the genus Zea (Terrell et al., 1986). Of the four species of Zea, only Z. mays is common in the United States. It is known only from cultivation; it occasionally is spontaneous in abandoned fields or roadsides, but is incapable of sustained reproduction outside of cultivation (Gould, 1968). The other species are occasional university or experiment station research subjects. Z. perennis is reported as established from James Island, South Carolina (Hitchcock and Chase, 1951).
The closest generic relative to Zea is Tripsacum, a genus of seven species, three of which occur in the United States (Gould, 1968). Tripsacum differs from corn in many respects, including chromosome number (n=9), in contrast to Zea (n=10). All species of Tripsacum can cross with Zea, but only with difficulty and all lead to sterile progeny (Galinat, 1988).
Cultivated corn is presumed to have been transformed from teosinte, Z. mays subspecies mexicana (Schrader) Iltis, more than 8000 years ago. During this transformation, cultivated corn gained several valuable agronomic traits, but lost the ability to survive in the wild. Teosinte, however, remains a successful wild grass in Mexico and Guatemala. Despite some confusion over proper taxonomic groupings of the non-cultivated members of Zea, the wild members of the genus, whether annual or perennial exhibit an array of visible chromosomal peculiarities and ploidy levels, and many adaptive macroscopic phenotypes. Cultivated corn and wild diploid and tetraploid members of Zea can be crossed to produce fertile F1 hybrids. Nonetheless, in the wild, introgressive hybridization does not occur because of differences in flowering time, geographic separation, block inheritance, developmental morphology, seed dissemination, and dormancy (Galinat, 1988).
The second major transformation of cultivated corn in the United States started in the 1930's and continues today through the development and use of inbred lines for hybrid seed production. Almost all corn grown in the United States now comes from hybrid seed that is obtained every planting season from private enterprises; the older open-pollinated varieties are now virtually unknown in commerce. This transformation has resulted in more uniform commercial plants with superior agronomic characteristics, and has contributed to the six-fold increase in yields over the last sixty years.
A variety of studies were conducted to characterize the unique traits of the modified corn line and to establish that insect protected corn line corn line MON 80100 is substantially equivalent to non-modified corn.
The CryIA(b) Protein
The CryIA(b) protein must be ingested by the insect to exert insecticidal activity (Huber and Lüthy, 1981). The protein in its crystalline form is insoluble in aqueous solution at neutral or acidic pH (Bulla et al, 1977); however, the alkaline pH of the larval insect gut dissolves the protein crystal. The dissolved protein is activated by proteases in the insect gut. The activated protein, approximately 600 amino acids long, diffuses through the peritrophic membrane of the insect to the midgut epithelium, binding to the specific high affinity receptors on the surface of the midgut epithelium of target insects (Wolfersberger et al, 1986; Hofmann et al, 1988a). The gut becomes paralyzed as a consequence of changes in electrolytes and pH gut causing the larval insect to quit feeding and die.
There are no receptors for the Bt protein delta-endotoxins on the surface of mammalian intestinal cells. Humans are therefore not susceptible to these proteins (Hofmann et al, 1988b; Noteborn, 1994; Sacchi et al, 1986) but digest them like other proteins. In addition to the lack of receptors for the Btk proteins, the absence of adverse effects in humans is further supported by numerous reviews on the safety of the Bt protein (Ignoffo, 1973; Shadduck, 1983; Siegel and Shadduck, 1989) and by rodent feeding (Naylor, 1992) and in vitro digestive fate studies of the Btk CryIA(b) protein (Ream, 1994).
Data were submitted to the EPA to support the registration and exemption from the requirement of a tolerance for the CryIA(b) protein as a plant pesticide. Studies included within that submission demonstrate the safety of this protein. In a mouse acute oral gavage study, no treatment related effects were observed in any of the groups of mice administered the CryIA(b) trypsin-resistant core protein by oral gavages at dosages up to 4000 mg/kg. The oral LD50 for the CryIA(b) protein in mice is greater than 4000 mg/kg and the no effect level is 4000 mg/kg (Naylor, 1992).
In an in vitro mammalian digestion study, the CryIA(b) protein degraded rapidly; more than 90% of the initially added CryIA(b) protein degraded after two minutes incubation in simulated gastric fluid as detected by western blot analysis and insect bioassay. In intestinal fluid, the trypsin-resistant core of the CryIA(b) protein did not degrade substantially after approximately 19.5 hours incubation as assessed by both western blot analysis and by insect bioassay (Ream, 1994). This result was expected as the trypsin-resistant core of this and other Bt insecticidal proteins have been shown to be relatively resistant to digestion by trypsin (Bielot et al, 1989). These results are fully consistent with the history of safe use of Bt preparations by humans.
The EPSPS Protein
EPSPS is an enzyme of the shikimate pathway for aromatic amino acid biosynthesis in plants (including corn) and microorganisms (Levin and Sprinson, 1964; Steinrüken and Amrhein, 1980), and is thus ordinarily present in food derived from plant sources. Genes for numerous EPSPS's have been cloned (Padgette et al, 1989, 1991), and active site domains are conserved among the known EPSPSs (Padgette et al, 1988, 1991). Bacterial EPSPSs have been well-characterized with respect to the 3-dimensional X-ray crystal structure (Stallings et al, 1991) and the chemical reaction mechanism (Anderson et al, 1990). EPSPSs from a number of bacteria exhibit tolerance to glyphosate (Schulz et al, 1985).
The herbicide glyphosate kills plants cells due to inhibition of the enzyme EPSPS (Steinrücken and Amrhein, 1980). The aromatic amino acid pathway is not present in mammalian metabolic pathways (Cole, 1985). This contributes to the selective action of glyphosate toward plants but not mammals. Glyphosate tolerance can be conferred to plant cells and microbes by either overproduction of EPSPS or the use of glyphosate-tolerant EPSPSs. The EPSPS from Agrobacterium sp. strain CP4 is highly tolerant to inhibition by glyphosate and has high catalytic efficiency, compared to most glyphosate-tolerant EPSPSs (Barry et al, 1992; Padgette et. al., 1991). Upon glyphosate treatment, the corn cells in the transformation process expressing the CP4 EPSPS are unaffected since the continued action of the glyphosate-tolerant EPSPS enzyme meets the plant's need for aromatic compounds.
CP4 EPSPS is a 47.6 kilodalton (KD) protein consisting of a single polypeptide of 455 amino acids. The gene encoding CP4 EPSPS has been completely sequenced. The enzyme has been expressed in E. coli and highly purified. CP4 EPSPS interacts with the EPSPS substrates shikimate-3-phosphate and phosphoenolpyruvate similarly to the plant enzymes, based on steady-state kinetic analyses. In addition, recent results indicate that the 3-dimensional X-ray crystal structure of CP4 EPSPS exhibits the same overall folding pattern as the E. coli EPSPS enzyme. The isolate CP4 was identified by the ATCC (American Type Culture Collection) as an Agrobacterium species, hence the designation Agrobacterium sp. strain CP4. Agrobacteria occur almost worldwide in soils and in the rhizosphere of plants. Agrobacterium strains have also been reported in a number of human clinical specimens, but it is believed that these clinical Agrobacterium isolates occur either as incidental inhabitants in the patient or as contaminants introduced during sample manipulation (Kersters and De Ley, 1984).
The chloroplast transit peptide (CTP) coding sequence from petunia EPSPS (Shah et al, 1986; Gasser et al, 1988) has been fused to the 5'-end of the CP4 EPSPS gene to deliver the CP4 EPSPS to the chloroplasts, the site of EPSPS activity and glyphosate action. Plant expression of the gene fusion produces a pre-protein which is rapidly imported into chloroplasts where the CTP is cleaved and degraded, releasing the mature CP4 EPSPS protein (della-Cioppa et al, 1986).
Expression Levels of the CryIA(b), CP4 EPSPS, GOX, and NPTII Proteins
Insect protected corn line MON 80100 has been modified to express a protein from Bacillus thuringiensis subsp. kurstaki strain CryIA(b) (Höfte and Whitely, 1989). This protein has insecticidal activity against the European corn borer (ECB), an economically damaging corn insect pest. In addition to the cryIA(b) gene, genes encoding CP4 EPSPS (Padgette et al, 1993), GOX (Padgette et al, 1994), and NPTII are also present. The CP4 EPSPS and GOX genes are present to enable selection of cells in tissue culture that contain the cryIA(b) gene. The corn transformation vectors used to produce corn line corn line MON 80100 included the gene cassette containing a bacterial specific promoter and coding region for NPTII. NPTII allows selection of bacteria containing the vector in media containing kanamycin. The nptII gene is under the control of a bacterial-specific promoter and, therefore, is not expected to produce the NPTII protein in plant cells.
A. The introduced genes, their products, and the added regulatory sequences do not present a plant pest risk in corn line MON 80100.
Two plasmids, PV-ZMBK07 and PV-ZMGT10, were transformed in corn genotype Hi-II by particle acceleration transformation system to generate corn line MON 80100.
The plasmid vector PV-ZMBK07 contains the cryIA(b) gene under the control of the enhanced 35S promoter (E35S) (Kay et al, 1985 and Odell et al, 1985), which is approximately 0.6 Kb in size. Located between the enhanced 35S promoter and the cryIA(b) gene is the 0.8 Kb intron from the hsp70 gene (heat-shock protein), present to increase the levels of gene transcription (Rochester et al, 1986). The cryIA(b) gene is joined to the 0.24 Kb nopaline synthase 3' nontranslated sequence, NOS 3', (Rogers et al, 1985) which provides the mRNA polyadenylation signals. The cryIA(b) gene is 3468 nucleotides in length and encodes a full-length Btk HD-1 [CryIA(b)] protein of 1156 amino acids, which when subjected to trypsin yields an active trypsin-resistant protein product of approximately 600 amino acids in planta and in vitro (Lee et al, 1995). The cryIA(b) gene sequence was modified to increase the levels of expression in corn using strategies similar to those as previously described (Perlak et al, 1991). The gene encodes the nature identical CryIA(b) Btk HD-1 protein product (Fischhoff et al, 1987). The alpha region of the lacZ gene for beta-galactosidase, present under a bacterial controlled promoter, is present in PV-ZMBK07. This region contained a polylinker (region with multiple cloning sites) which allowed for the cloning of the desired genes within the plasmid vector (Vieira and Messing, 1987). Most of the approximately 200 bp region of the alpha region of the gene for beta-galactosidase was not incorporated into corn line corn line MON 80100. The lacZ-alpha region is followed by the 0.7 Kb origin of replication for the pUC plasmids (ori-pUC) and which allows for the replication of plasmids in E. coli (Vieira and Messing, 1987). Following the ori-pUC region is the gene for the enzyme neomycin phosphotransferase type II (NPTII). This enzyme confers resistance to aminoglycoside antibiotics (i.e., kanamycin and neomycin) and was used for selection of bacteria during the construction of this plasmid. The coding sequence for the nptII gene was derived from the prokaryotic transposon Tn5 (Beck et al, 1982) and is present under its own bacterial promoter. The NPTII protein was not detected in corn line corn line MON 80100 (Ream et al, 1995).
The PV-ZMGT10 plasmid contains the gox and CP4 EPSPS genes joined to chloroplast transit peptides CTP1 and CTP2, respectively. Both coding regions are under the control of the enhanced 35S promoter, hsp70 intron and NOS 3' terminator sequences. The
PV-ZMGT10 vector contains the same lacZ-alpha, ori-pUC and nptII regions as described above for PV-ZMBK07. The CP4 EPSPS and gox genes enable the selection of cells in tissue culture that contain the cryIA(b) gene. A CP4 EPSPS has been isolated from Agrobacterium sp. strain CP4 which has been shown to be highly resistant to glyphosate (Harrison et al, 1993). The CP4 EPSPS protein represents one of many different EPSPSs found in nature (Schulz et al, 1985) and is highly tolerant to the inhibition by glyphosate and has high catalytic efficiency, compared to most EPSPSs (Barry et al, 1992; Padgette et al, 1991). Upon glyphosate treatment, plants or plant cells expressing the CP4 EPSPS protein are unaffected since the continued action of the tolerant EPSPS enzyme meets the plant's need for aromatic compounds. The CP4 EPSPS gene in PV-ZMGT10 contains a chloroplast transit peptide, CTP2, isolated from Arabidopsis thaliana EPSPS (Klee and Rogers, 1987) which directs the CP4 EPSPS protein to the chloroplast, the location of EPSPS in plants and the site of aromatic amino acid synthesis (Kishore and Shah, 1988). The CP4 EPSPS gene with its CTP2 is approximately 1.7 Kb in size. The CP4 EPSPS gene cassette (promoter through 3' termination sequence) is joined to the gox cassette.
The gox gene that encodes the glyphosate metabolizing enzyme glyphosate oxidoreductase (GOX) was cloned from Achromobacter sp. strain LBAA (Hallas et al, 1988; Barry et al, 1992; Barry et al, 1994). The GOX protein is targeted to the plastids with a chloroplast transit peptide sequence, CTP1. The CTP1 was derived from the small subunit gene of ribulose-1,5-bisphosphate carboxylase (SSU1A) gene from Arabidopsis thaliana (Timko et al, 1988). The enzyme GOX degrades glyphosate by converting glyphosate to aminomethylphosphonic acid (AMPA) and glyoxylate. The GOX protein was not detected by ELISA or western blot analysis in the seeds or leaves of corn line MON 80100.
The plant produced EPSPSs are present in the chloroplast. Therefore, the chloroplast transit peptide coding sequence, CTP2, from Arabidopsis thaliana EPSPS (Klee et al, 1987) was fused to the N-terminus of the CP4 EPSPS protein to deliver the CP4 EPSPS to the chloroplasts, the site of EPSPS activity and glyphosate action (Kishore and Shah, 1988). CTPs are typically cleaved from the ?mature" protein following delivery to the plastid (della Cioppa et al, 1986). The bacterial selectable marker gene, nptII , isolated from the prokaryotic transposon, Tn5 (Beck et al, 1982), encodes for the enzyme neomycin phosphotransferase II which confers resistance to the aminoglycoside antibiotics (i.e., kanamycin or neomycin) used for selection of plasmids in E. coli. The promoter for this gene is only active in bacterial hosts. As expected, no NPTII protein was detected in the insect protected corn line corn line MON 80100 using western blot analysis.
B. Expression of insect control protein in the corn line MON 80100 will not likely provide a competitive advantage sufficient to cause these plants to become any more "weedy" than other corn.
APHIS evaluated whether the corn line MON 80100 is any more likely to become a successful weed than nontransgenic control corn. Most definitions of weediness stress the undesirable nature of weeds from the point of view of humans; individual definitions differ in approach and emphasis (Baker, 1965; Muenscher, 1980). Baker defines a plant as a weed if, in any specified geographical area, its populations grow entirely or predominantly in situations markedly disturbed by man (without, of course, being deliberately cultivated) (Baker, 1965). He also described the ideal characteristics of weeds as including the following: discontinuous germination and long-lived seeds; rapid seedling growth; rapid growth to reproductive stage; long continuous seed production; self-compatibility, but not obligatory self-pollination or apomyxis; if outcrossing, use of wind or an unspecialized pollinator; high seed output under favorable conditions; germination and seed production under a wide range of environmental conditions; high tolerance or plasticity of climatic and edaphic variation; special adaptations for dispersal; good competitiveness achieved through, for example, allelochemicals or choking growth; and, if perennial, then exhibiting vigorous vegetative reproduction, brittleness either at the lower nodes or of rhizomes or rootstocks, and having the ability to regenerate from severed rootstocks. Although Baker's characteristics have been criticized by some ecologists as nonpredictive, no more broadly accepted suite of characteristics has been defined by ecologists (Williamson, 1994). In our view, there is no formulation that is clearly superior at this time. Keeler (1989) and Tiedje et al. (1989) have adapted and analyzed Baker's list to develop admittedly imperfect guides to the weediness potential of transgenic plants. Both authors emphasize the importance of looking at the parent plant and the nature of the specific genetic changes. Cultivated corn, particularly the line CG00526 variety, lacks most of Baker's "weedy" characteristics (Keeler, 1989). Corn is not listed as a common, serious or principal weed or a weed of current or potential importance in the United States and/or Canada in most weed compendiums (Holm et al., 1991; Muenscher, 1955; USDA, 1971; Weed Science Society of America, 1992).
Expression of the insect control protein in the corn line MON 80100 will not likely provide a competitive advantage sufficient to cause these to be any more "weedy" than other corn cultivars. None of the characteristics of weeds described by Baker involved resistance or susceptibility to insects. More importantly, in addition to the analysis above, APHIS evaluated field data submitted by Monsanto Company which demonstrate that corn line MON 80100 is no more weedy than any other corn. Control and corn line MON 80100 plants were routinely monitored during field trials for differences in morphological characteristics, disease and insect susceptibility.
Based on evaluation of the available literature and data submitted by Monsanto Company, APHIS concludes that the corn line MON 80100 is no more likely to present a plant pest risk as a weed than are nontransgenic controls.
C. Gene introgression from corn line MON 80100 into wild or cultivated sexually-compatible plants is unlikely, and such rare events should not increase the weediness potential of resulting progeny or adversely impact biodiversity.
APHIS evaluated the potential for gene flow from corn line MON 80100 to other cultivated and wild relatives. Then two potential impacts that might result from this sexual transfer of genes were evaluated: first, that the traits from corn line MON 80100 might cause free-living relatives to become "weedier", and second, that the transfer of genes might cause population changes that would lead to reduced genetic diversity. The glyphosate resistance trait used as a selectable marker in the corn line MON 80100 was considered not to pose a hazard in this analysis. Glyphosate is already registered for use in corn in special application manner.
Gene introgression into other corn cultivars via cross pollination is possible. If pollen of the corn line MON 80100 was transferred by wind or any other means to receptive corn stigma within the 30 minute period of pollen viability, cross-pollination could occur. The potential for such transfer decreases as distance increases from the transgenic plants. From a practical standpoint, it becomes increasingly unlikely at distances much beyond the foundation seed isolation distance of 660 feet. Farmers purchase hybrid corn seed for planting from a commercial source. If pollen of corn line MON 80100 fertilizes corn in the fields of a farmer, any resulting corn seed will likely be used for some thing other than seed (eg oil or meal). Fertilization of nontransgenic corn by pollen from corn line MON 80100 in fields grown for sale as food or feed will not result in dissemination of the trait in to seed populations used for replanting. Moreover, seeds resulting from such fertilization will not themselves express the introduced genetic material.
As stated in Section IV., Monsanto Company reported no obvious differences in the flowering of corn line MON 80100 compared to the nontransgenic parent. There is no reason to believe that the genetic construct introduced during the transformation event would have any effect on the reproductive biology of the corn line MON 80100, unless the insertion event interrupted a genetic locus critical for the normal reproductive function.
Breeder seed is usually derived from self-pollinated seed at around the eighth to tenth generations of inbreeding (Wych, 1988). A high degree of self-pollination is ensured by planting well isolated blocks that virtually guarantee random mating. Minimum isolation distances for foundation seed are one-eighth mile (660 feet) from the nearest contaminating source. Other safeguards, such as physical barriers or border trap rows, can further reduce the possibility of contamination. Fields are preferred that have not been recently planted in corn. This is to minimize the appearance of volunteer corn from a previous season. Corn appears as a volunteer in some fields and roadsides, but it does not become established outside of cultivation (Gould et al, 1994).
D. Use of corn line MON 80100 should have no more adverse impacts on raw or processed agricultural commodities than the parent corn.
During extensive field testing, the corn line MON 80100 exhibited the typical agronomic characteristics of the recipient, with the exception of the desired corn line MON 80100 phenotype conferred by the Btk insect control protein. In APHIS' opinion, the components, quality and processing characteristics of corn line MON 80100 reveal no differences that could have an indirect plant pest effect on any raw or processed plant commodity. None of the sequences introduced is associated with any disease specific property of the donor organism from which it was derived, and corn line MON 80100 exhibit no plant pest characteristics.
E. Corn line MON 80100 exhibits no significant potential to harm organisms beneficial to agriculture, to harm threatened or endangered organisms or to have an adverse impact on the ability to control nontarget insect pests.
Organisms beneficial to the agricultural ecosystem: Consistent with its statutory authority and requirements under NEPA, APHIS evaluated the potential for corn line MON 80100 plants and plant products and the Btk insect control protein to have damaging or toxic effects directly or indirectly on nontarget organisms, particularly those that are recognized as beneficial to agriculture. APHIS also considered potential impacts on other nontarget pests, since such impacts could have an impact on the potential for changes in agricultural practices. There is no reason to believe that the EPSPS and GOX protein conferring glyphosate resistance in the corn line MON 80100 plants as a selectable marker for transformation would have deleterious effects or significant impacts on nontarget organisms, including beneficial organisms. Glyphosate herbicide, the target of the EPSPS gene product is not registered for use in corn. There have been no reports of toxic effects on such organisms in the many field trials conducted with many different plants expressing these selectable markers.
Data and information supplied by the applicant demonstrate that corn line MON 80100 is substantially equivalent to non-modified corn, except for the inserted genetic sequences, the expressed proteins [CryIA(b) protein, GOX and CP4 EPSPS enzymes], and the resulting ability of the plant to resist damage from certain Lepidopteran insects including European corn borer. Corn line MON 80100 is not likely to pose a greater plant pest risk than non-modified corn. This conclusion is based on evaluation of phenotypic characteristics, safety of the inserted proteins, and the lack of any deleterious environmental effects.
1. Non-target Insects
There is extensive information on the lack of non-target effects from microbial preparations of Bacillus thuringiensis subsp. kurstaki (Btk) containing the Btk proteins, including the CryIA(b) protein. The literature has established that the Btk proteins: are highly specific for lepidopteran insects (MacIntosh et al, 1990; Klausner, 1984; Aronson et al, 1986; Dulmage, 1981; Whitely and Schnepf, 1986). They bind specifically to receptors on the mid-gut of lepidopteran insects (Wolfersberger et al, 1986; Hofmann et al, 1988a; Hofmann et al, 1988b; Van Rie et al, 1989; Van Rie et al, 1990); and have no deleterious effect on beneficial/non-target insects, including predators and parasitoids of lepidopteran insect pests or honeybees (Apis mellifera) (Flexner et al, 1986; Krieg and Langenbruch, 1981; Cantwell et al, 1972; EPA, 1988; Vinson, 1989; Melin and Cozzi, 1989).
The chapters by Vinson (1989) and Melin and Cozzi (1989) provide comprehensive reviews of the extensive literature that has established the safety of the Btk microbes and encoded proteins to an array of beneficial insects. In addition, separate studies were undertaken to assess the potential toxicity of the CryIA(b) protein to other non-target insects.
a. Honey bee larvae and adults
These studies assessed the potential toxicity of the CryIA(b) trypsin-resistant core protein to larvae and adult honey bee (Apis mellifera L.), a beneficial insect pollinator. The maximum nominal CryIA(b) protein concentration tested was greater than 10 times the estimated LC50 sensitivity of several target pest Lepidoptera to the CryIA(b) protein. The LC50 for the CryIA(b) protein in larval and adult honey bee is greater than 20 ppm. The no observed effect level was 20 ppm (Maggi and Sims, 1994a, 1994b).
b. Green lacewing
This study assessed the potential toxicity of the CryIA(b) trypsin-resistant core protein to green lacewing larvae (Chrysopa carnea), a beneficial predaceous insect commonly found in corn and other cultivated plants. There was no evidence that green lacewing larvae were adversely effected when fed moth eggs coated with a nominal concentration of 16.7 ppm CryIA(b) protein for seven days. Under the conditions of the test, the LC50 was greater than 16.7 ppm CryIA(b) protein (Hoxter and Lynn, 1992a).
c. Parasitic hymenoptera
This study assessed the potential toxicity of the CryIA(b) trypsin-resistant core protein to parasitic Hymenoptera (Brachymeria intermedia), a beneficial parasite of the housefly (Musca domestica). Parasitic Hymenoptera exposed to activated CryIA(b) protein at a concentration of 20 ppm in honey/water solution for thirty days did not exhibit treatment related mortality or signs of toxicity. The LC50 for CryIA(b) protein in parasitic Hymenoptera is greater than 20 ppm. The no-observed effect level was 20 ppm (Hoxter and Lynn, 1992c).
d. Ladybird beetles
This study evaluated the potential toxicity of CryIA(b) trypsin-resistant core protein to ladybird beetles (Hippodamia convergens), a beneficial predaceous insect which feeds on aphids and other plant insects commonly found on stems and foliage of weeds and cultivated plants. Ladybird beetles exposed to activated CryIA(b) protein at a test concentration of 20 ppm in a honey/water solution for nine days did not exhibit treatment related mortality or signs of toxicity. The LC50 for CryIA(b) protein in ladybird beetles is greater than 20 ppm. The no-observed effect level was 20 ppm (Hoxter and Lynn, 1992b).
Insect protected corn line MON 80100 also encodes the enzyme CP4 EPSPS as discussed above. EPSPS is an enzyme of the shikimate pathway for aromatic amino acid biosynthesis in plants (including corn) and microorganisms (Levin and Sprinson, 1964), and is thus ordinarily present in food derived from plant sources. Genes for numerous EPSPS's have been cloned (Padgette et al, 1989, 1991), and active site domains are conserved among the known EPSPSs (Padgette et al, 1988, 1991). Bacterial EPSPSs have been well-characterized with respect to the 3-dimensional X-ray crystal structure (Stallings et al, 1991) and the detailed kinetic and chemical reaction mechanism (Anderson et al, 1990). EPSPSs from a number of bacteria exhibit tolerance to glyphosate (Schulz et al, 1985). CP4 EPSPS thus represents one of many different EPSPSs found in nature. EPSPS is considered to be ubiquitous in nature since it is present in all plants and microorganisms. Therefore, all organisms which presently feed on plants and/or microbes, historically have been exposed to EPSPS.
2. Non-Target Wildlife and Fish
A study was conducted to assess the wholesomeness of insect protected corn meal fed to quail since birds may feed on corn left in the field after harvest. No mortality occurred in birds fed up to 10% w/w (nominal 100,000 ppm) raw corn seed meal in the diet. This feeding level approximates consumption of 138 corn seeds/kg body weight/ bird/day. The no-observed effect level was considered to be greater than 10% w/w. Based on the parameters measured, the wholesomeness of meal from insect protected corn seed was comparable to that of the parental line when fed in the diet to quail (Campbell and Beavers, 1994).
It is unlikely that fish in their natural environment would be exposed to corn seed. Based on the historical data demonstrating the safety of Bt proteins to fish and the unlikely event of exposure, no adverse effects are expected to fish from the use of the CryIA(b) protein as expressed in corn.
3. Impact on Endangered Species
No endangered or threatened lepidopteran insects, as listed in 50CFR 17.11 and 17.12, feed on corn plants.
Environmental Fate of the CryIA(b) Protein
Previous work has demonstrated the rapid loss of insecticidal activity of Bacillus thuringiensis protein crystals (active ingredient) derived from microbial preparations of lepidopteran-active species when incubated in the soil (Palm et al, 1994; Pruett et al, 1980; West, 1984). The CryIA(b) protein in insect protected corn line MON 80100 is present at low levels in the plant tissue remaining in the field after the harvest of the seed or silage. This corn plant material may be tilled into the soil or remain on the soil surface as is typically observed in zero tillage systems. The environmental fate of the CryIA(b) protein was determined by measuring the rate at which the bioactivity of the CryIA(b) protein dissipated when added to soil as the purified protein and as a component of insect protected corn tissue.
Studies examined the dissipation rate of the CryIA(b) protein from three systems: 1) insect protected corn tissue without contact with soil, 2) insect protected corn tissue mixed into soil, and 3) purified CryIA(b) protein mixed into soil. The levels incorporated into the soil were greater than three fold higher than the maximum concentration expected under field conditions. CryIA(b) protein, added to soil as a component of tissue from insect protected corn had an estimated DT50 of 1.6 days. Bioactivity of insect protected corn tissue, incubated without soil contact, had an estimated DT50 of 25.6 days. Purified CryIA(b) protein, mixed into the soil, had as estimated DT50 of 8.3 days. This rate of dissipation of insecticidal activity is comparable to that observed with microbial Bt products.
Therefore, results of this study suggest that the CryIA(b) protein, as a component of post-harvest insect protected corn plants, will dissipate readily on the surface of (e.g. no-till), or when cultivated into the soil. The measured half-life of the purified Btk protein in soil is comparable to that measured for the microbial Btk preparations (Palm et al, 1994; Pruett et al, 1980; West, 1984).
Endangered lepidopterans may conceivably be sensitive to CryIA(b) protein, given that the protein is selectively toxic to certain lepidopteran species. Endangered lepidopterans include several species of moths and butterflies: the El Segundo blue butterfly, the primrose sphinx moth, Lange's metalmark butterfly, the Lotis blue butterfly, the Oregon silverspot butterfly, the San Bruno elfin butterfly, Schaus' swallowtail butterfly, and Smith's blue butterfly. These species are not found in areas where corn is commonly grown. Corn is not among the host plants for these lepidopterans. Unlike the exposure scenarios typical of conventional or microbial insecticides, an organism must actually consume maize tissue to receive any exposure to CryIA(b). Therefore, we conclude that threatened and endangered organisms will not be affected as a result of corn line MON 80100.
Host-range comparisons have not indicated any change in range of species susceptible to maize CryIA(b) compared to native CryIA(b). In its effects on insects, corn line MON 80100 is similar to the microbial insecticidal preparations that are already commercially sold. Both field testing and laboratory testing of maize CryIA(b) have indicated that nontarget beneficial insects are not likely to be affected by maize CryIA(b), so it is not likely that endangered dipterans, hymenopterans, or coleopterans would be affected.
Ability to control nontarget insect pests: In all of the studies outlined above, there is no evidence that exposure to the CryIA(b) protein expressed in either maize pollen or extracted from Bt maize leaves resulted in any toxic effect on the organism tested. Tested organisms include representative avian, aquatic invertebrates, and soil invertebrate species, and several nontarget insect species. Testing with insects known to be either susceptible or not susceptible to native CryIA(b) gave no indications of a changed host specificity for the maize expressed CryIA(b). A small field monitoring study on beneficial insects did not detect any effects on beneficial insect or insect prey species exposed to Bt maize. These results suggest that only lepidopterans susceptible to native CryIA(b) are likely to be affected by Bt maize CryIA(b).
APHIS concludes that corn line MON 80100 exhibit no significant potential to adversely impact organisms beneficial to plants or agriculture or to adversely impact the ability to control nontarget insect pests of agriculture.
F. Cultivation of corn line MON 80100 should not reduce the ability to control insects in corn and other crops.
APHIS considered potential impacts associated with the cultivation of corn line MON 80100 on the current agricultural practices used to control insects. Monsanto Company also provided APHIS a copy of their strategy for maximizing the utility of these plants and delaying the development of insect-protected to the Btk insect control protein. The development of effective resistance management strategies is an ongoing process already submitted by the petitioner for EPA consideration, and APHIS will continue to offer comments and recommendations for technical improvements to the EPA and Monsanto Company to assist in this process. The EPA has stated that they are committed to working with Monsanto Company to develop product labels and informational brochures that include instructions on the proper use of the corn line MON 80100 that are consistent with resistance management.
Both chemical and microbial insecticides are currently used for control of ECB in corn. Among chemical insecticides organophosphates (Counter®, Dyfonate®, Lorsban®, Thimet®, Parathion®, Penncap®), pyrethroids (Ambush®, Pounce®, Capture®), carbamate (Furadan®) and others (Asana XL®) are used. Though organophosphates and pyrethroids can be effective against ECB, careful insect surveillance is required. Applications must be carefully timed to reach insect populations before the insects bore into the stalk, and repeated applications are often necessary.
B. thuringiensis var. kurstaki preparations are registered for use on corn, vegetables, cotton, deciduous nuts and fruits. As crystalline powder formulations, Btk has been used commercially as an insecticide under the trade name Dipel. Only 5% of the commercially grown corn is treated with Btk preparations to control ECB. ECB reduces corn yields by causing physical damage to the plant and ear that result in weakness of the stalk dropped ears, and damaged grain. Yield reductions due to ECB infestation are estimated to exceed $50 million annually in the State of Illinois alone. Btk is very effective in the laboratory against ECB. However, commercial Btk formulations are generally ineffective in controlling ECB on corn because topical applications of the powder do not reach the inside the corn stalks where the insects feed. Ciba has engineered the corn line MON 80100 to produce the Btk insecticide specifically in corn tissue where ECB feeds. The use of this corn should provide farmers a means of controlling a serious insect pest that is not easily controlled by current chemical pesticides. Other advantages include: (1) reducing the risks associated with environmental spills or misapplication of chemical insecticides; (2) eliminating unwanted effects on beneficial insect populations (which can be susceptible to conventional chemical applications); these beneficial insects can, in turn, further reduce the reliance upon chemical means of pest control; and (3) reducing the consumption of fossil fuels required to deliver chemical inputs by machinery.
Corn line MON 80100 could provide a more effective alternative for season-long control of ECB compared to the use of foliar microbial Btk products that currently only receive limited use. Monsanto Company field data indicate that corn line MON 80100 is more effective than some Btk formulations at reducing insect survival and egg-laying. The corn line MON 80100 alternative is particularly important where chemical insecticides are no longer effective due to insect resistance. Over the course of use of corn line MON 80100, resistant insects will probably evolve but to recommend a resistance management strategy is outside the scope of this analysis.
Monsanto Company transgenic corn line MON 80100 offers an alternative control method. Monsanto Company strategies for maximizing the utility of these plants while delaying the development of insect resistance to these plants include the following:
1. Promoting the incorporation of corn line MON 80100 into integrated pest management programs (IPM) that emphasize the use of cultural control practices, such as those described above, and judicious and selective use of additional insecticides only when pest populations reach the threshold for economic damage. They do not encourage the use of corn line MON 80100 as a stand-alone control measure.
2. Monitoring insect populations for Btk protein susceptibility so that development of resistance can be detected and management strategies altered accordingly.
3. High dose expression of Btk protein to control insect that are heterozygous for resistance alleles.
4. Deployment of other corn lines or other hosts as refugia for insects that are sensitive to the Btk insect control protein, in order to maintain susceptible alleles in the population.
5. Development of new insect control proteins with a distinct mode of action to be employed with the Btk protein.
6. Implementation of a grower education program to achieve items 1, 2, and 4 above.
APHIS evaluated the potential impact to agricultural practices associated with the use of corn line MON 80100 according to Monsanto Company strategy. As a result of Monsanto Company program to instruct growers on the use of cultural control practices and IPM, growers may be more likely to adopt these methods. However, growers will also need to be informed about the implementation of preferred refugia strategies and how these can be integrated with other cultural practices. Growers will be less likely to use chemical insecticides targeted at insect control, and this should reduce the risks associated with some of these insecticides. The use of corn line MON 80100 should increase safety to field workers and consumers, reduce toxicity to nontarget species, and lower rates of ground water contamination by insecticides. Corn line MON 80100 plants are not likely to eliminate completely the use of chemical insecticides, particularly when they may be needed to control other serious pests. But perhaps they may encourage more selective use of insecticides against these pests.
Monsanto Company support of research in resistance development to corn line MON 80100 and their grower education plan, coupled with ECB population monitoring programs, could enable them to implement strategies to delay resistance and detect and possibly contain it when it occurs. It may be possible to control resistant ECB populations by the use of agronomic practices such as crop rotation and alternate insecticides.
APHIS concludes that development of resistance to insecticides is a potential associated with their use; but in this respect, cultivation of corn line MON 80100 should pose no greater effects on the control of insects in corn and other crops, than the widely practiced method of applying insecticides.
V.CONCLUSION
APHIS has determined that corn line MON 80100 and its progeny will no longer be considered regulated articles under APHIS regulations at 7 CFR Part 340. Permits or notifications acknowledged under those regulations will no longer be required from APHIS for field testing, importation, or interstate movement of those corn line MON 80100 or their progeny. (Importation of corn line MON 80100 seeds capable of propagation is still, however, subject to the restrictions found in the Foreign Quarantine Notice regulations at 7 CFR Part 319.) This determination has been made based on data collected from these trials, laboratory analyses and literature references presented herein which demonstrate the following:
1. Corn line MON 80100 exhibits no plant pathogenic properties. Although pathogenic organisms were used in their development, these corn plants are not infected by these organisms nor can these plants incite disease in other plants.
2. Corn line MON 80100 is no more likely to become a weed than insect-protected corn which could potentially be developed by traditional breeding techniques. Corn is not a serious, principal or common weed pest in the U.S., and there is no reason to believe that resistance to insects would enable corn to become weed pests.
3. Multiple barriers insure that gene introgression from Corn line MON 80100 into wild or cultivated sexually-compatible plants is extremely unlikely, and such rare events should not increase the weediness potential of any resulting progeny or adversely impact biodiversity.
4. Seeds of corn line MON 80100 are substantially equivalent in composition, quality and other characteristics to nontransgenic corn and should have no adverse impacts on raw or processed agricultural commodities.
5. Corn line MON 80100 exhibits no significant potential either to harm organisms beneficial to the agriculture or to impair the ability of farmers to control nontarget insect pests.
6. Cultivation of corn line MON 80100 should not reduce the ability to control insects in corn and other crops.
APHIS has also concluded that there will be new varieties bred from corn line MON 80100; however, if such varieties were developed they are unlikely to exhibit new plant pest properties, i.e., properties substantially different from any observed for corn line MON 80100 already field tested, or those observed for corn developed from traditional breeding.
John H. Payne, Ph.D. Date
Acting Director
Biotechnology, Biologics, and Environmental Protection
Animal and Plant Health Inspection Service
U.S. Department of Agriculture
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