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Monsanto Petition 95-045-01p to USDA/APHIS for Determination of Nonregulated Status of Glyphosate Tolerant Cotton (Roundup Ready^TM) lines 1445 and 1698

Environmental Assessment and

Finding of No Significant Impact

July 1995

The Animal and Plant Health Inspection Service (APHIS) of the U. S. Department of Agriculture has prepared an environmental assessment before issuing a determination of nonregulated status for genetically engineered cotton known as glyphosate tolerant cotton (Roundup Ready^TM) lines 1445 and 1698. APHIS received a petition from Monsanto Company regarding the status of transformation lines 1445 and 1698 as regulated articles under APHIS regulations at 7 CFR Part 340. APHIS has conducted an extensive review of the petition and supporting documentation, as well as other relevant scientific information. Based on the analysis documented in this environmental assessment, APHIS has reached a finding of no significant impact on the environment from its determination that glyphosate tolerant cotton (Roundup Ready^TM) lines 1445 and 1698 shall no longer be regulated articles.

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John H. Payne, Ph.D.

Acting Director

Biotechnology, Biologics, and Environmental Protection

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

Date:

I. SUMMARY

The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA), has prepared an Environmental Assessment (EA) to decide whether or not the glyphosate tolerant (Roundup Ready^TM) cotton lines 1445 and 1698 (hereafter referred to as lines 1445 and 1698) should continue to be a regulated article under 7 CFR 340. Monsanto Company (hereafter referred to as Monsanto) petitioned APHIS requesting a determination on 1445 and 1698, and any progeny derived from them, as non-regulated articles under APHIS regulations found at 7 CFR Part 340 (hereafter referred to as the regulations). The petition contained information pertinent to the company claim that lines 1445 and 1698 do not present a plant pest risk and therefore, should no longer be regulated articles under the APHIS regulations. Lines 1445 and 1698 have been considered regulated articles because they were engineered with DNA sequences derived from the plant pathogenic sources. As regulated articles, APHIS permits and notifications have been required for introductions (importation, interstate movements, and field tests) of 1445 and 1698.

Lines 1445 and 1698 were developed by using recombinant DNA techniques to introduce a gene for EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), isolated from Agrobacterium sp. strain CP4 that encodes an enzyme which is naturally tolerant to glyphosate, the active ingredient of Roundup® herbicide. In addition, the nptII (neomycin phosphotransferase II) gene isolated from the bacterial transposon Tn5 that codes for resistance to the antibiotic kanamycin to function as a selectable marker in plants has been introduced into lines 1445 and 1698. The transgenic cotton lines that are subject of the petition were developed by a widely used technique called Agro-infection which essentially involves using a plant pathogenic strain of Agrobacterium tumefaciens and its disarmed plasmid vector.

Roundup® herbicide contains the active ingredient glyphosate which is a non-selective, post-emergent weed control agent. The target site of action of glyphosate is EPSPS that is present in all plants, bacteria, and fungi as a component of the Shikimate pathway of aromatic amino acid biosynthesis (Levin and Sprinson, 1994). The CP4 EPSPS which is naturally resistant to the inhibition by glyphosate (Padgette et al. 1993) has been introduced into 1445 and 1698 to confer tolerance to the foliar application of glyphosate.

Separate EAs were prepared by APHIS before granting the permits for every field trial with 1445 and 1698. Previous EAs (91-347-01, and 93-012-02, and 93-012-03 have addressed questions pertinent to plant pest risk issues relevant to the conduct of field trials under physical and reproductive confinement, but they do not address several issues that are relevant to the unconfined cultivation, and notifications 93-223-02n, 93-210-02n, 94-027-02n, and 94-273-03n were approved based on those EAs. With respect to these new issues, APHIS concludes the following:

1. Lines 1445 and 1698 exhibit no plant pathogenic properties. Although DNA sequences from a plant pathogen were used in their development, these cotton plants are not infected nor can these plants incite disease in other plants.

2. Lines 1445 and 1698 are no more likely to become weeds than cotton developed by traditional breeding techniques. Cotton is not considered to be a serious, principal or common weed pest in the U.S.

3. Lines 1445 and 1698 are unlikely to increase the weediness potential for any other cultivated or wild species with which they can interbreed. The introgression of the EPSPS gene from 1445 and 1698 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. Lines 1445 and 1698 will not harm other organisms, including agriculturally beneficial organisms and threatened and endangered species.

5. Lines 1445 and 1698 should not cause damage to processed agricultural commodities. Seeds of 1445 and 1698 are substantially equivalent in composition, quality, and other characteristics to nontransgenic cotton plants and should have no adverse impacts on raw or processed agricultural commodities.

Therefore, after a review of the available evidence, APHIS believes that 1445 and 1698 will be just as safe to grow as traditionally bred cotton varieties that are not subject to APHIS regulation under 7 CFR Part 340. APHIS concludes that there will be no significant impact on the human environment if 1445 and 1698 or their progeny were no longer considered regulated articles under the regulations.

II. BACKGROUND

Development of 1445 and 1698. In a petition dated February 9, 1995 Monsanto Company requested a determination from APHIS that 1445 and 1698, and any progeny derived from them, should no longer be considered regulated articles under APHIS regulations found at 7 CFR Part 340. 1445 and 1698 have been considered regulated articles because they were engineered with DNA sequences derived from the plant pathogenic sources, and the transformation employed Agrobacterium tumefaciens.

Transgenic cotton lines 1445 and 1698 were developed by using recombinant DNA techniques to introduce a gene for EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), isolated from Agrobacterium sp. strain CP4 that encodes an enzyme which is naturally tolerant to glyphosate, the active ingredient of Roundup® herbicide. In addition, nptII (neomycin phosphotransferase II) gene isolated from bacterial transposon Tn5 that codes for resistance to the antibiotic kanamycin to function as a selectable marker in plants, and another selectable marker gene aad (3"(9)-O-aminoglycoside adenylyltransferase (AAD) to function in bacterial cells to confer resistance to streptomycin and spectinomycin antibiotics have also been introduced into lines 1445 and 1698, but not expressed. The transgenic cotton lines that are the subject of the petition were developed by the use of a plant pathogenic strain of Agrobacterium tumefaciens and its disarmed plasmid vector.

Roundup herbicide contains the active ingredient glyphosate which is a non-selective, post-emergent weed control agent. The target site of action of glyphosate is EPSPS that is present in all plants, bacteria, and fungi as a component of the Shikimate pathway of aromatic amino acid biosynthesis (Levin and Sprinson, 1994). The CP4 EPSPS which is naturally resistant to the inhibition by glyphosate (Padgette et al. 1993) has been introduced into 1445 and 1698 to confer tolerance to the foliar application of glyphosate.

1445 and 1698 have been field tested since 1991 in the major cotton growing regions of the United States under permits and acknowledgements of notifications by APHIS (USDA Permit Numbers

91-347-01 in Alabama, and 93-012-02 in Alabama, Arkansas, Arizona, California, Georgia, Louisiana, Mississippi, and Texas, 93-012-02 in North Carolina, and notifications 93-223-02n and 93-210-02n in Puerto Rico, 94-027-01n in Alabama, Arkansas, Arizona, California, Georgia, Louisiana, Mississippi, Tennessee, and Texas, 94-027-02n in North Carolina, and 94-273-03n in Alabama, Louisiana, and Texas). Lines 1445 and 1698 have been evaluated extensively in laboratory, greenhouse, and field experiments to confirm that they exhibit the desired agronomic characteristics and do not present a plant pest risk. Although the field tests of 1445 and 1698 have been conducted in agricultural settings, the permit conditions and acknowledgement of notifications for the tests have stipulated physical and reproductive confinement from other plants.

APHIS Regulatory Authority. APHIS regulations at 7 CFR Part 340, which were promulgated pursuant to authority granted by the Federal Plant Pest Act, (7 U.S.C. 150aa-150jj) as amended, and the Plant Quarantine Act, (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.

A genetically engineered organism is considered a regulated article if the donor organism, recipient organism, vector or vector agent used in engineering the organism belongs to one of the taxa listed in the regulation and is also a plant pest, or if there is reason to believe that it is a plant pest.

Section 340.6 of the regulations, entitled "Petition Process for Determination of Nonregulated Status", provides that a person may petition APHIS to evaluate submitted information and determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article is unlikely to pose a greater plant pest risk than the unmodified organism, APHIS can grant the petition in whole or in part. Therefore, APHIS permits would no longer be required for field testing, importation, or interstate movement of that article or its progeny.

Environmental Protection Agency (EPA) and Food and Drug Administration (FDA) Regulatory Authority. 1445 and 1698 are also subject to regulation by other agencies. APHIS' decision on the regulatory status of 1445 and 1698 under APHIS' regulations at 7 CFR 340, does not release this cotton and its progeny from EPA and FDA regulatory oversight. The EPA is responsible for the regulation of pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136 et seq.). Therefore, any use of herbicides on 1445 and 1698 will be regulated by EPA. FDA's policy statement concerning regulation of products derived from new plant varieties, including those genetically engineered, was published in the Federal Register on May 29, 1992, and appears at 57 FR 22984-23005.

III. PURPOSE AND NEED

APHIS has prepared this EA before determining the status of 1445 and 1698 as regulated articles under APHIS regulations. The developer of 1445 and 1698, Monsanto Company, submitted a petition to APHIS requesting that APHIS make a determination that 1445 and 1698 and their progeny shall no longer be considered regulated articles under APHIS regulations (7 CFR Part 340).

This EA was prepared in compliance with the National Environmental Policy Act (NEPA) of 1969 (40 CFR 1500-1508) and the pursuant implementing regulations published by the Council on Environmental Quality (42 USC 4331 et seq.; 40 CFR 1500-1508; 7 CFR Part 1b; 44 FR 50381-50384; and 44 FR 51272-51274).

IV. ALTERNATIVES

A. No Action.

Under the Federal "no action" alternative, APHIS would not come to a determination that 1445 and 1698 are no longer regulated articles under the regulations at 7 CFR Part 340. Permits from APHIS would still be required for introductions of 1445 and 1698. APHIS might choose this alternative if there were insufficient evidence to demonstrate the lack of plant pest risk from uncontained cultivation of 1445 and 1698.

B. Determination that 1445 and 1698 is no longer a regulated article.

Under this alternative, 1445 and 1698 would no longer be regulated articles under the regulations at 7 CFR Part 340. Permits from APHIS would no longer be required for introductions of 1445 and 1698. A basis for this determination would include a "Finding of No Significant Impact" under the NEPA.

V. AFFECTED ENVIRONMENT AND POTENTIAL ENVIRONMENTAL IMPACTS

This EA addresses potential environmental impacts from an APHIS determination that 1445 and 1698 should no longer be considered regulated articles. Previous EAs (91-347-01, and 93-012-02, and 93-012-03 have addressed questions pertinent to plant pest risk issues relevant to the conduct of field trials under physical and reproductive confinement, but they do not address several issues that are relevant to the unconfined cultivation, and notifications 93-223-02n, 93-210-02n, 94-027-02n, and 94-273-03n were approved based on those EAs. This EA discusses the genetic modifications and the potential environmental impacts that might be associated with the unconfined cultivation of 1445 and 1698.

Additional technical information is included in the determination document appended to this EA, and is incorporated by reference. The determination includes more detailed discussions of the biology of cotton, the genetic components used in the construction of 1445 and 1698, and the analyses that lead APHIS to conclude that 1445 and 1698 have no potential to present a plant pest risk.

A. Potential for the introduced genes, their products, and the added regulatory sequences controlling their expression to present a plant pest risk in transformed lines 1445 and 1698

Transgenic cotton lines 1445 and 1698 were developed by using recombinant DNA techniques to introduce a gene for EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), isolated from Agrobacterium sp. strain CP4 that encodes an enzyme which is naturally tolerant to glyphosate, the active ingredient of Roundup® herbicide. In addition, nptII (neomycin phosphotransferase II) gene isolated from bacterial transposon Tn5 that codes for resistance to the antibiotic kanamycin to function as a selectable marker in plants, and another selectable marker gene aad (3"(9)-O-aminoglycoside adenylyltransferase (AAD) to function in bacterial cells to confer resistance to streptomycin and spectinomycin antibiotics but does not express have also been introduced into lines 1445 and 1698. The transgenic cotton lines that are the subject of the petition were developed by a widely used technique called Agro-infection which essentially involves using a plant pathogenic strain of Agrobacterium tumefaciens and its disarmed plasmid vector.

Roundup® herbicide contains the active ingredient glyphosate which is a non-selective, post-emergent weed control agent. The target site of action of glyphosate is EPSPS that is present in all plants, bacteria, and fungi as a component of the Shikimate pathway of aromatic amino acid biosynthesis (Levin and Sprinson, 1994). The CP4 EPSPS which is naturally resistant to the inhibition by glyphosate (Padgette et al. 1993) has been introduced into 1445 and 1698 to confer tolerance to the foliar application of glyphosate.

It is amply clear from our previous EAs and also from the scientific evidence in the published literature that even though several DNA components and live bacteria were used to develop these transgenic cotton lines, none of these components from plant pathogenic organisms have any inherent ability to incite disease in the transformed plants as they have either been disarmed or the bacteria have been killed with a potent antibiotic. As such they do not present a risk of plant pests being introduced into the environment by way of uncontained cultivation of these transgenic cotton lines 1445 and 1698.

B. Potential for 1445 and 1698 to become successful weeds

Cotton has been grown for centuries throughout the world without any reports that it is a serious weed pest, and it is unlikely to become a weed pest. In the United States, cotton is not listed as a weed in the major weed references (Crockett 1977; Holm ^{et al}. 1979; Muenscher 1980), nor is it present on the lists of noxious weed species distributed by the Federal Government (7 CFR Part 360).

The parent plant of 1445 and 1698 is a line of cotton (Gossypium hirsutum L.) known as Coker 312 that exhibits no appreciable weedy characteristics. The EPSPS gene is unlikely to increase weediness of 1445 and 1698. The glyphosate resistance of these plants will confer a selective advantage only when glyphosate is applied to the plants. No other attributes of 1445 and 1698 suggest that it be any more "weedy" than traditionally-bred cotton cultivars. Other than the resistance to the herbicide glyphosate, 1445 and 1698 have retained the agronomic characteristics of the parental cotton, including the sensitivity to other herbicides.

Monsanto Company has provided data regarding seed germination rates, yield characteristics, disease and pest susceptibilities, compositional analyses, and numerous other tests which support APHIS' conclusion that 1445 and 1698 are no more likely to become weeds than cotton developed by traditional breeding techniques.

C. Potential for 1445 and 1698 to increase the weediness potential of any other plant with which it can interbreed.

Cotton belongs to the genus Gossypium of the tribe Gossypeae of the family Malvaceae (Fryxell, 1979; Munro, 1987). Only four species of cotton are of any agronomic importance in the world; two diploid old world cotton or Asiatic cotton and two allotetraploid New World species. The old world cotton is restricted to India, Africa and Asia (Munro, 1987). But, the new world cotton comprises of 98% of cotton cultivated for fibre production. Wild species of cotton occur in arid parts of the tropics and subtropics. Fryxell (1984) has divided the wild diploid species into three geographical groups: the Australian group (11 species), the Afro-Arabian group (8 species), and the American group (12 species). Two species of the American group (wild tetraploid) occur in Peru and in the Galapagos, and the remaining 10 occur in Western Mexico with one (G. thurberi Todaro) extending up to Arizona. G. tomentosum and G. hirsutum are two of the new world cottons that occur in Hawaii and middle America and drier areas of southern tip of Florida (Fryxell, 1984; Lee, 194). Wild populations of G. hirsutum are relatively rare and tend to be widely dispersed as beach strands or on small islands. There are examples of escaped cotton belonging to G. hirsutum and G. barbedense growing in the wild in Southern Florida and Hawaii. These escaped plants appear opportunistic toward disturbed land and appear not be effective in inhabiting managed ecosystems.

Although natural outcrossing can occur in cotton, it is normally self-pollinating (Niles and Feaster, 1984). The pollen is heavy and sticky, and is heavily pollinated via bumble bees and honey bees. The range of natural crossing is very limited (100-200 feet) (McGregor, 1976).

APHIS considered whether the movement of the EPSPS gene from 1445 and 1698 to other cultivated cotton or wild relatives might result in offspring that would present problems as weeds. The genetic integrity of commercial cultivated cotton lines and varieties is strictly controlled through established plant breeding practices. These standard practices make it unlikely that this glyphosate tolerance trait will be inadvertently incorporated into the germplasm of cultivated cotton lines.

D. Potential for 1445 and 1698 to harm other organisms, including agriculturally beneficial organisms and threatened or endangered species.

Consistent with its statutory authority and requirements under NEPA, APHIS evaluated the potential for 1445 and 1698 to directly or indirectly harm other organisms, including those that are recognized as beneficial to agriculture and to those that are recognized as threatened or endangered in the United States.

APHIS concluded that the available evidence suggests that 1445 and 1698 will not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and will not harm threatened or endangered species.

The use of glyphosate herbicides in the cultivation of 1445 and 1698, or their offspring will be regulated by the EPA under its existing regulations for the registration of pesticide use. As part of the pesticide registration process, EPA considers the impacts on the environment, including organisms.

E. Potential for 1445 and 1698 to damage agricultural commodities.

APHIS can envision no way in which 1445 and 1698 would damage agricultural commodities. With the exception of two enzymes, EPSPS and NPTII, the composition and attributes of 1445 and 1698 are indistinguishable from the parental line of cotton used to develop 1445 and 1698. There is no indication that the EPSPS enzyme itself will affect the qualities of commodities derived from 1445 and 1698.

VI. CONCLUSION

APHIS has evaluated information from the scientific literature as well as information submitted by Monsanto Company that characterized 1445 and 1698. After careful analysis, APHIS has identified no significant impact to the environment from issuance of a determination that 1445 and 1698 should no longer be regulated articles under APHIS regulations at 7 CFR Part 340. This finding is supported by the following conclusions:

2. Lines 1445 and 1698 are no more likely to become weeds than cotton developed by traditional breeding techniques. Cotton is not considered to be a serious, principal or common weed pest in the U.S.

4. Lines 1445 and 1698 will not harm other organisms, including agriculturally beneficial organisms and threatened and endangered species.

5. Lines 1445 and 1698 should not cause damage to processed agricultural commodities. Seeds of 1445 and 1698 are substantially equivalent in composition, quality, and other characteristics to nontransgenic yellow dent cotton and should have no adverse impacts on raw or processed agricultural commodities.

Therefore, after review of the available evidence, APHIS concludes that 1445 and 1698 will be just as safe to grow as traditionally-bred cotton varieties that are not subject to regulation under 7 CFR Part 340. APHIS concludes that there should be no significant impact on the human environment if 1445 and 1698 were no longer considered regulated articles under its regulations at 7 CFR Part 340.

VII. LITERATURE CITED

Crockett, L. 1977. Wildly Successful Plants: North American Weeds. University of Hawaii Press, Honolulu, Hawaii. 609 pp.

Fryxell, P. A. 1979. Natural history of the cotton tribe (Malvaceae, tribe Gossypeae), Texas A&M Press, College Station, Texas.

Fryxell, P. A. 1984. Cotton, Agronomy No. 24, pp-82-129, Soil Science Society of America, Inc. (Kohel, R. J. and C. F. Lewis, eds.), Wisconsin, USA.

Fryxell, P. A. 1984. Cotton, Agronomy No. 24, pp-82-129, Lee, J. A. 1984. Cotton, Agronomy No. 24, p.25, Soil Science Society of America, Inc. (Kohel, R. J. and C. F. Lewis, eds.), Wisconsin, USA.

Holm, L., Pancho, J.V., Herbarger, J.P., Plucknett, D.L. 1979. A Geographical Atlas of World Weeds. John Wiley and Sons, New York. 391 pp.

McGregor, S. E. 1976. Insect pollination of cultivated crop plants. Agricultural Handbook No. 496. United States Department of Agriculture, Agricultural Research Service, Washington, D.C.

Muenscher, W. C. 1980. Weeds. Second Edition. Cornell University Press, Ithaca and London. 586 pp.

Munro, J. M. 1987. Cotton, Second Edition. John Wiley & Sons, New York, NY.

Padgette, S. R., Q. K. Huynh, S. Aykent, R. D. Sammons, J. A. Sikorski, and G. M. Kishore. 1988. Identification of the reactive cysteines of Eshcrichia coli 5-enolpyruvylshikimate-3-phosphate synthase and their nonessentiality for enzymatic catalysis. J. Biol. Chem. 263:1798-1802.

Padgette. S. R., Q. K. Hyunh, J. Borgmeyer, D. M. Shah, L. A. Brand, D. B. Re, B. F. Bishop, S. G. Rogers, R. T. Fraley, and G. M. Kishore. 1987. Bacterial expression and isolation of Petunia hybrida 5-enolpyruvyl-shikimate-3-phospahte synthase. Arch. Biochem. Biophys. 258:564-573.

Padgette, S. R., G. F. Barry, D. B. Re, M. Weldon, D. A. Eiccholtz, K. H. Kolacz, R. Heeren, B. Bishop, and G. M. Kishore. 1994. Purification, cloning, and characterization of a highly glyphosate-tolerant EPSP synthase from Agrobacterium sp. strain CP4. Manuscript in Prparation).

VIII. PREPARERS AND REVIEWERS

Biotechnology, Biologics, and Environmental Protection (BBEP)

Terry L. Medley, J.D., Director

(Acting Associate Administrator, APHIS)

John Payne, Ph.D., Associate Director

(Acting Director, BBEP)

Biotechnology Permits

Arnold Foudin, Ph.D., Deputy Director

Subhash Gupta, Ph.D., Biotechnologist

David S. Heron, Ph.D., Biotechnologist

Susan Koehler, Ph.D., Biotechnologist

James Lackey, Ph.D., Biological Safety Officer

Vedpal Malik, Ph.D., Biotechnologist

H. Keith Reding, Ph.D., Biotechnologist

Sivramiah Shantharam, Ph.D., Chief, Microorganisms Branch (Chief Preparer)

James L. White, Ph.D., Chief, Plants Branch

Biotechnology Coordination and Technical Assistance

Michael A. Lidsky, J.D., LL.M., Deputy Director

L. Val Giddings, Ph.D., Chief, Science Policy & Coordination Branch

Shirley P. Ingebritsen, M.A., Program Analyst

Michael Schechtman, Ph.D., Senior Microbiologist

Frank Y. Tang, Ph.D., J.D., Biotechnologist

Quentin K. Kubicek, Ph.D., Senior Biotechnologist

Environmental Analysis and Documentation

Carl Bausch, J.D., Deputy Director

IX. AGENCY CONTACT

Ms. Kay Peterson, Regulatory Analyst

Biotechnology, Biologics, and Environmental Protection

USDA, APHIS

4700 River Road, Unit 147

Riverdale, MD 20737-1237

Phone: (301) 734-7612

Fax: (301) 734-8669