Page 1 of 85 August 20, 2004 MEMORANDUM SUBJECT: Transmittal of Minutes of the FIFRA Scientific Advisory Panel Meeting Held June 8 10, 2004: Product Characterization, Human Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis ( Bt) Cotton Products TO: James J. Jones, Director Office of Pesticide Programs FROM: Paul I. Lewis, Designated Federal Official FIFRA Scientific Advisory Panel Office of Science Coordination and Policy THRU: Larry C. Dorsey, Executive Secretary FIFRA Scientific Advisory Panel Office of Science Coordination and Policy Joseph J. Merenda, Jr., Director Office of Science Coordination and Policy Please find attached the minutes of the FIFRA Scientific Advisory Panel open meeting held in Arlington, Virginia from June 8 10, 2004. These meeting minutes address a set of scientific issues being considered by the U. S. Environmental Protection Agency regarding Product Characterization, Human Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis ( Bt) Cotton Products Attachment Page 2 of 85 cc:

Susan Hazen Margaret Schneider Adam Sharp Anne Lindsay Janet Andersen Debbie Edwards Steven Bradbury William Diamond Arnold Layne Tina Levine Lois Rossi Frank Sanders Margaret Stasikowski William Jordan Douglas Parsons Karen Chu Dayton Eckerson Enesta Jones Vanessa Vu ( SAB) Leonard Cole John Kough Zigfridas Vaituzis Sharlene Matten OPP Docket Page 3 of 85 FIFRA SAP Members Gary Isom, Ph. D. Janice Elaine Chambers, Ph. D. H. Christopher Frey, Ph. D. Kenneth Portier, Ph. D. Mary Anna Thrall, D. V. M.

FQPA Science Review Board Members Jonathan A. Arias, Ph. D. Bruce Chassy, Ph. D. Steven Gendel, Ph. D. Dr. Alexander Haslberger Barrie Kitto, Ph. D. Luke Masson, Ph. D. George Cobb, Ph. D. Paul Jepson, Ph. D. Thomas La Point, Ph. D. Steven Naranjo, Ph. D. Michael C. Newman, Ph. D. Sean Richards, Ph. D. Janice Thies, Ph. D. Michael Caprio, Ph. D. Steven Castle, Ph. D. Jesus Esquivel, Ph. D. Steven L. Peck, Ph. D. Richard Roush, Ph. D. John Schneider, Ph. D. Douglas Sumerford, Ph. D. Mark Whalon, Ph. D. Page 4 of 85 SAP Report No. 2004 05 MEETING MINUTES FIFRA Scientific Advisory Panel Meeting, June 8 10, 2004, held at the Holiday Inn Arlington , Arlington, Virginia A Set of Scientific Issues Being Considered by the U. S. Environmental Protection Agency Regarding:

Product Characterization, Human Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis ( Bt) Cotton Products Page 5 of 85 NOTICE These meeting minutes have been written as part of the activities of the Federal Insecticide, Fungicide, and Rodenticide Act ( FIFRA), Scientific Advisory Panel ( SAP). This report has not been reviewed for approval by the United States Environmental Protection Agency ( Agency) and, hence, the contents of this report do not necessarily represent the views and policies of the Agency, nor of other agencies in the Executive Branch of the Federal government, nor does mention of trade names or commercial products constitute a recommendation for use.

The FIFRA SAP was established under the provisions of FIFRA, as amended by the Food Quality Protection Act ( FQPA) of 1996, to provide advice, information, and recommendations to the Agency Administrator on pesticides and pesticide related issues regarding the impact of regulatory actions on health and the environment. The Panel serves as the primary scientific peer review mechanism of the EPA, Office of Pesticide Programs ( OPP) and is structured to provide balanced expert assessment of pesticide and pesticide related matters facing the Agency. Food Quality Protection Act Science Review Board members serve the FIFRA SAP on an ad hoc basis to assist in reviews conducted by the FIFRA SAP. Further information about FIFRA SAP reports and activities can be obtained from its website at http:// www. epa. gov/ scipoly/ sap/ or the OPP Docket at ( 703) 305 5805. Interested persons are invited to contact Paul Lewis, Designated Federal Official, via e mail at lewis. paul@ epa. gov.

In preparing these meeting minutes, the Panel carefully considered all information provided and presented by the Agency presenters, as well as information presented by public commenters. This document addresses the information provided and presented within the structure of the charge by the Agency. Page 6 of 85 SAP Report No. 2004 05 MEETING MINUTES:

FIFRA Scientific Advisory Panel Meeting, June 8 10, 2004, held at the Holiday Inn Arlington, Arlington, Virginia A Set of Scientific Issues Being Considered by the U. S. Environmental Protection Agency Regarding:

Product Characterization, Human Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis ( Bt) Cotton Products Mr. Paul Lewis Gary Isom, Ph. D. Designated Federal Official FIFRA SAP Session Chair FIFRA Scientific Advisory Panel FIFRA Scientific Advisory Panel Date: August 17, 2004 Date: August 17, 2004 Page 7 of 85 Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel Meeting June 8 10, 2004 Product Characterization, Human Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis ( Bt) Cotton Products PARTICIPANTS FIFRA SAP Session Chair Gary Isom, Ph. D., Professor of Toxicology, School of Pharmacy & Pharmacal Sciences, Purdue University, West Lafayette, IN FIFRA Scientific Advisory Panel Members Janice Elaine Chambers, Ph. D., William L. Giles Distinguished Professor and Director, Center for Environmental Health Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS H. Christopher Frey, Ph. D., Associate Professor, Civil Engineering, North Carolina State University, Raleigh, NC Kenneth Portier, Ph. D., Associate Professor, Statistics, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL Mary Anna Thrall, D. V. M., Professor, Microbiology, Immunology & Pathology, Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Fort Collins, CO FQPA Science Review Board Members Jonathan A. Arias, Ph. D., Associate Research Scientist, Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD Michael Caprio, Ph. D., Associate Professor, Insect Genetics, Entomology & Plant Pathology, Mississippi State University, Mississippi State, MS Steven Castle, Ph. D., Research Entomologist, USDA/ ARS, Western Cotton Research Laboratory, Phoenix, AZ Bruce Chassy, Ph. D., Associate Executive Director, Campus Biotechnology Center and Professor, Department of Food Science and Human Nutrition, University of Illinois, Urbana, IL George Cobb, Ph. D., Associate Professor, Environmental Toxicology, Texas Tech University, Lubbock, TX Page 8 of 85 Jesus Esquivel, Ph. D., Research Entomologist, Areawide Pest Management Research Unit, USDA/ ARS SPARC, College Station, TX Steven Gendel, Ph. D., Branch Chief, Food and Drug Administration, National Center for Food Safety and Technology, Summit Argo, IL Paul Jepson, Ph. D., Director, Integrated Plant Protection Center, Oregon State University, Corvallis, OR Dr. Alexander Haslberger, Professor of Microbiology, Institute of Microbiology and Genetics, University of Vienna, Vienna, Austria Barrie Kitto, Ph. D., Professor of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX Thomas La Point, Ph. D., Professor and Director, Biological Sciences and Institute of Applied Sciences, University of North Texas, Denton, TX Luke Masson, Ph. D., Senior Research Scientist, National Research Council of Canada, Biotechnology Research Institute, Montreal, Quebec, Canada Steven Naranjo, Ph. D., Research Entomologist, USDA/ ARS, Western Cotton Research Laboratory, Phoenix, AZ Michael C. Newman, Ph. D., Professor of Marine Science, Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, VA Steven L. Peck, Ph. D., Assistant Professor, Department of Integrative Biology, Brigham Young University, Provo, UT Sean Richards, Ph. D., UC Foundation Assistant Professor, Department of Biological and Environmental Sciences, University of Tennessee at Chattanooga, Chattanooga, TN Richard Roush, Ph. D., Director, Statewide IPM Program, University of California, Davis, CA John Schneider, Ph. D., Professor and Research Entomologist, Department of Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS Douglas Sumerford, Ph. D., Research Entomologist, Corn Insects and Crop Genetics Research Unit, Genetics Laboratory c/ o Insectary, USDA/ ARS, Iowa State University, Ames, IA Janice Thies, Ph. D., Associate Professor, Soil Biology, Department of Crop and Soil Science, Cornell University, Ithaca, NY Page 9 of 85 Mark Whalon, Ph. D., Professor, Center for Integrated Plant Systems, Michigan State University, East Lansing, MI PUBLIC COMMENTERS Oral statements were made by: Rod Herman, Ph. D., Dow Agrosciences Monte Mayes, Ph. D., Dow Agrosciences Larry Sernyk, Ph. D., Dow Agrosciences Nick Storer, Ph. D., Dow Agrosciences Laura Tagliani, Ph. D., Dow Agrosciences Ray Layton, Ph. D., Dupont Company, on behalf of the Agricultural Biotechnology Stewardship Technical Committee, Non target Organism Subcommittee Jane Rissler, Ph. D., Union of Concerned Scientists Graham Head, Ph. D., Monsanto Written statements were received from: National Cotton Council Monsanto JR Bradley, North Carolina State University INTRODUCTION The Federal Insecticide, Fungicide, and Rodenticide Act ( FIFRA), Scientific Advisory Panel ( SAP) has completed its review of the set of scientific issues being considered by the Agency pertaining to its review of product characterization, human health risk, ecological risk, and insect resistance management for Bacillus thuringiensis ( Bt) cotton products. Advance notice of the meeting was published in the Federal Register on May 12, 2004. The review was conducted in an open Panel meeting held in Arlington, Virginia, from June 8 10, 2004. The meeting was chaired by Gary Isom, Ph. D. Mr. Paul Lewis served as the Designated Federal Official. Mr. Joseph J. Merenda, Jr. ( Director, Office of Science Coordination and Policy, EPA) and Janet Andersen, Ph. D. ( Director, Biopesticides and Pollution Prevention Division, Office of Pesticide Programs, EPA) offered opening remarks at the meeting. Mr. Leonard Cole ( Office of Pesticide Programs, EPA) provided an introduction and highlighted the goals and objectives of the meeting. The Agency's product characterization and human health safety assessment for stacked plant incorporated protectants, environmental effects assessment for WideStrike cotton, and issues related to establishing an insect resistance management plan for WideStrike cotton were presented by John Kough, Ph. D. ( Office of Pesticide Programs, EPA), Zigfridas Vaituzis, Ph. D. ( Office of Pesticide Programs, EPA) and Sharlene Matten, Ph. D. ( Office of Pesticide Programs, EPA), respectively. Sharlene Matten, Ph. D. ( Office of Pesticide Programs, EPA) concluded the Agency's presentations by discussing Bollgard and Bollgard II cotton bollworm insect resistance management. Page 10 of 85 SUMMARY OF PANEL DISCUSSION AND RECOMMENDATIONS Human Health Risk The Panel concluded that toxicity studies showed no adverse effects at the highest levels tested, either for the individual Cry proteins or for the two proteins together. The Panel agreed with the Agency that combined oral toxicity studies for multiple proteins is not necessary if each protein had been previously independently tested, unless such testing would be intended to detect any effects produced by interactions between the proteins.

Based on the evidence presented in the safety assessment of the single Bt containing strain and the more recent extensive testing of the stacked Cry1F/ Cry1Ac material, the Panel concluded that there is little to suggest that the stacked variety poses significantly more risk or unanticipated consequences to agronomic performance than do varieties with either the Cry1F or Cry1Ac genes introduced singly.

Ecological Risk Direct hazards to vertebrates due to exposure to the Cry1F and Cry1Ac proteins should be minimal or non existent, making consideration of synergy in vertebrates unwarranted. Thus, the Panel concluded that toxicity testing on the combinations of Cry proteins is not necessary. While synergism is not considered to be important for the Cry1 proteins under consideration, the Panel believed that future testing of non target effects of these types of toxins should proceed, with both toxins to be expressed by genetically engineered plants rather than with individual purified toxins. Testing mixtures of compounds would provide data that are more relevant to the proposed use scenarios and likely exposure scenarios.

The Panel recognized improvements in the quality of the Agency's analysis and the degree to which this reflected a positive response by the Agency to recommendations made by previous SAP reviews of plant incorporated protectants ( PIPs). The Panel also noted that the Tier I tests were generally of a higher quality than the tests that other SAPs have reviewed. Overall, however, detailed analysis of the field based evaluations revealed that these did not meet current standards, and the Panel repeated requests made by other SAPs, that the Agency issue detailed guidelines for semi field, and field based procedures. The Panel recommended that the exposure pathway and diet used within the Chrysoperla test be examined further, to determine the rigor and repeatability of the test, and also suggested that Orius spp. be considered as a more appropriate test subject. The Panel also considered that a more appropriate parasitoid than Nasonia should be selected.

The Panel agreed that multi year field studies to assess potential longer term effects of WideStrike cultivation on persistence of toxins in the soil and on populations of non target organisms are applicable to Bt cotton and should be considered. Overall, the likely reduction in the use of broader spectrum insecticides afforded by the cultivation of Bt cotton in general is likely to have positive effects on the ecosystems. Nonetheless, longer term and broader scale Page 11 of 85 evaluation of PIP crops will be necessary for improved ecological understanding. The Panel believed strongly that long term field studies should not proceed without some guidance relative to experimental protocols and clearly defined endpoints. Without such guidance, we are unlikely to resolve any unexpected detriments or benefits associated with the use of transgenic crops.

WideStrike Cotton Insect Resistance Management The combined expression of Bt proteins in WideStrike cotton meets the Agency's definitions of high dose for pink bollworm ( PBW) and tobacco budworm ( TBW). In addition, reasonable doses of the combined protein were evident for control of cotton bollworm ( CBW). Based on the high dose evidence, the Panel concluded that it is valid to assume that resistance occurring in PBW or TBW will likely be inherited as a recessive trait. However, CBW is more tolerant of both proteins and it seems possible that resistance will be less recessive. WideStrike cotton does appear to offer a high dose for TBW, a high dose of Cry1Ac for PBW, and reasonable doses of Cry1F and Cry1Ac for CBW. The same high dose/ refuge strategy practiced thus far as a resistance management approach for Bollgard cotton should be applied for WideStrike.

While the Panel supported the Agency's conclusion that incomplete shared binding of Cry1Ac and Cry1F receptors in TBW and CBW is expected to lead to incomplete crossresistance differences were expressed on the molecular mechanism involved in the process. In addition the Panel raised the issue that another as yet unidentified major resistance mechanism may not occur. The Panel agreed with the Agency that there is no basis to believe that the occurrence of resistance in the field will be due to a mechanism other than binding site modification.

The Panel identified several areas of concern with the Dow Agrosciences CBW model that make its use problematic. These problems must be addressed if this model is to be used to assess the durability of WideStrike cotton. The Panel believed that use of the current model, once corrected of the identified errors, would be an appropriate vehicle to explore the parameter space with the goal of finding areas where resistance does occur in the 15 year time horizon and assessing whether it occurs within biologically plausible initial conditions and parameter values.

Since the dose of the Cry1Ac and Cry1F in WideStrike Cotton was demonstrated to be high against populations of TBW, the Panel believed that WideStrike will be more durable than that predicted by Peck ( 1999) for single Cry1Ac cotton.

The Panel agreed that the HOSTS database is insufficient to address the issue of CBW alternate hosts as natural refugia. The Panel agreed that there are insufficient empirical data in the registrant report to demonstrate that alternative hosts are producing susceptible, fit individuals in sufficient quantity, at the correct time and proximity to maximize the probability of matings between homozygous susceptible individuals and individuals heterozygous for resistant traits.

Even though the Panel raised limitations with the model as described, the Panel was in Page 12 of 85 strong agreement that the proposed IRM plan by the registrant is sufficient for WideStrike cotton and supported the prediction of a delay in resistance of TBW, CBW and PBW to WideStrike cotton for 15 years. The overall consensus was that the existing IRM options that have been applied to the single toxin Bollgard cotton will be equally or even more effective in protecting against resistance in the double toxin WideStrike cotton.

Bollgard Insect Resistance Management The Panel could not determine whether CBW reverse migration is expected to have any impact on CBW adaptation to Bt cotton or Bt corn.

While the Panel agreed that pyrethroid oversprays in Bollgard cotton improve the control of susceptible corn earworm ( CEW), the effect of pyrethroid oversprays in delaying resistance in CEW is probably overstated. The Panel agreed that there is little need to include pyrethroid oversprays in Bollgard II plots in the models of Gustafson et al. There is some evidence of greater tolerance in larvae originating from Bollgard fields relative to those coming from non Bollgard fields.

The Panel agreed that sufficient data were provided to establish that C3 and C4 alternate hosts function to some degree as unstructured refugia. However, the Panel expressed concern on the methodologies used to assess adult productivity in the alternate hosts. The Panel agreed that CBW production should be measured at a larger scale than the local farm, or field level because of the high mobility of adult CBW. In response to the request for methods on quantitatively calculating " effective refuge size," the Panel provided techniques for quantifying CBW populations in the identified alternate hosts that were identified as natural refugia.

The Panel agreed with the Agency that a weighted average is an appropriate choice for determining the contribution of alternate hosts to the refuge size. The Panel believed, however, that exploring detailed questions about time to resistance and the effect of alternate hosts on resistance would benefit from the development of a more detailed model.

PANEL DELIBERATIONS AND RESPONSE TO THE CHARGE The specific issues to be addressed by the Panel are keyed to the Agency's background documents, references and Agency's charge questions.

Agency Charge Human Health Risk 1. The Agency examines the safety of proteins based on the source of the protein, the protein's pesticidal mode of action, comparisons of the amino acid sequence to toxins and allergens and the results of acute oral toxicity testing. The company provided numerous mammalian oral toxicity studies to demonstrate the safety of the introduced Cry 1Ac and Page 13 of 85 Cry1F protein insecticidal toxins. The toxins were tested both separately and in combination. The Agency believes tests with combinations of pure proteins may address possible synergistic interactions between introduced proteins. However, the Agency believes that unless there is an indication that the two proteins would interact, such as being parts of a binary toxin or attaching to the same receptor, there is little to justify testing the two proteins together when separate oral toxicity tests indicate a lack of toxicity for the individual proteins.

Does the Panel have additional comments on this position including identifying instances where it would be justified to require the toxicity testing of two proteins in combination?

Panel Response The Panel's comments to this question are specific to expressed proteins. Comments on unintended effects are presented by the Panel in their response to question 2. The relevant human health issue is related to consumption of these proteins. A search of the literature regarding these substances failed to find any indication that there are any other significant issues directly related to human health.

The Agency has been asked to evaluate a line of cotton containing two Bt Cry proteins intended for insect control. This line was produced by cross breeding two lines containing independent transformation events. The pesticide registrant provided data characterizing the two transformation events, showing that the structures of the inserts were not altered during crossing, and demonstrating stable Mendelian inheritance of each insert. In terms of human health, the pesticide registrant provided sequence analysis data for all the proteins involved ( the Cry proteins and the markers), stability data, and the results of oral toxicity studies using mice. The Panel concluded that these toxicity studies showed no adverse effects at the highest levels tested, either for the individual Cry proteins or for the two proteins together. It should be noted that the testing was done with material that was highly equivalent ( but not identical) to the proteins expressed in plants. In addition, because cotton proteins make only a very low contribution to the human diet, exposure to these proteins is expected to be very low.

The fact that the cotton line involved expressed two insect control proteins raised the question of whether, or when, it is necessary to carry out combined oral toxicity studies for multiple proteins if each protein has been independently tested previously. This testing would be intended to detect any effects produced by interactions between the proteins. The Agency has stated the belief that such testing is not justified unless there is specific evidence for such interactions. In general, the Panel agreed with this statement.

The basic principles for assessing the safety of transferred proteins in bioengineered plants have been well developed. A good summary of these principles can be found in the CODEX document " Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant DNA Plants." In general, these principles suggest that, for proteins, safety assessment should focus on source, function, similarity to known toxins, anti nutrients and Page 14 of 85 allergens, and stability. This guidance suggested that " appropriate oral toxicity studies may need to be carried out in cases where the protein   is not similar to proteins have that previously been consumed safely." These are essentially the same criteria specified by the Agency.

First, previous evaluations of potential human health risks associated with ( PIPs) plantincorporated protectant proteins may be considered as having set a precedent for this approach. In earlier cases where only a single PIP protein was expressed in a transformed plant, it was in fact accompanied by a second protein that was not active insecticidally, but instead served as a marker to identify a transformation event. In the present consideration of WideStrike cotton, synthetic genes coding for the insecticidal proteins Cry1F and Cry1Ac were integrated independently into separate cotton breeding lines and accompanied, in both cases, by the pat gene encoding for the marker protein phosphinothricin acetyltransferase ( PAT). In effect, the individual transformation events for each PIP protein involved expression of two new proteins in a target plant   the marker and the pesticidal protein. In these cases, each protein was evaluated independently. The classical breeding of the two transformed cotton lines eventually produced WideStrike cotton that expresses the pyramided PIP proteins Cry1Ac and Cry1F along with the intact marker protein PAT and an incomplete version of PAT. Whereas the insert from the Cry1Ac event ( 3006 210 23) contains one intact copy of the insect resistance gene cry1Ac and one intact copy of the plant selectable marker gene pat, the insert from the Cry1F event ( 281 24 236) contains one intact copy of the insect resistance gene Cry1Ac, one intact copy of the marker gene pat, plus an additional hybridizing fragment of the marker gene pat ( Dow AgroSciences Study 010075.01). The presence of the partial pat fragment results in a 4th product expressed in WideStrike cotton that has a potential amino acid sequence 90% identical to PAT and generates the same homology profile as the full length PAT sequence ( Dow AgroSciences Study ID: GH C 5573). Despite its truncated resemblance to the intact PAT protein and reduced expression, the 4th product must still be considered along with Cry1Ac, Cry1F and PAT for potential effects on human health. The question remains whether testing of two or more proteins together is necessary when separate toxicity tests for individual proteins indicate a lack of toxicity. Since the Panel agreed with the Agency that testing for interactions between the proteins is not justified unless specific evidence points to the contrary, the Panel sees no basis why, if independent evaluation is considered appropriate for two proteins, it should not also be considered appropriate for four or more proteins.

Second, although there are many examples of binary ( or multimeric) protein toxins, the interactions between the proteins involved are specific among members of a toxin family. The Panel is not aware of any instances where a " new" toxin has been created by unexpected interaction between two known proteins. Conversely, it seems unlikely that two proteins that interact to produce a biological effect would independently have unexpected activity, except in well defined situations ( for example as blockers of transport or digestion pathways). Again, it appears unlikely that unexpected interactions will occur between two unrelated proteins. The only examples would be if physical interactions between two unstable proteins inhibited gastric digestion or increased stability during processing. Therefore, it is reasonable to treat each protein independently in situations such as this. It is possible that simple biochemical tests could be used to determine whether such physical interactions occur between independent proteins, in which Page 15 of 85 case the combination can be targeted for digestion studies.

Third, the principle of independent assessment has been used for many years for food additives and is implicit in microbial risk assessments. There is no reason to believe that the issues involved with these proteins should affect the use of this principle.

Fourth, the relatively low expression levels for the proteins involved make it unlikely that they would be able to interact extensively and maintain their biological function. Unanticipated interactions seem more likely to result in sequestration and reduced activity for the proteins involved.

Fifth, given that each of these proteins has previously been used independently in food plants, the Panel concluded that they would fit the criteria of being similar to proteins that have previously been consumed safely. Therefore, they do not rise to the level of concern that would suggest the need for combined toxicity testing.

In the charge to the Panel, the Agency also raised the possibility that having two proteins attached to the same receptor could trigger a need for combined toxicity testing. It appears that, in this context, this means binding to the same receptor in humans. Any proteins that have known biological effects in humans need to be evaluated, regardless of mechanism.

There is one point of terminology that needs clarification by the Agency  the definition of the term " stacked" and " pyramidal" such as when used for stacked traits or stacked proteins. There are different ways to " stack" or " pyramid" traits ( multiple transformation or cross breeding) and it is unclear to the Panel whether the Agency considers these differences to be significant.

Agency Charge 2. When traits are introduced into crop plants using the transformation techniques of modern biotechnology or even traditional breeding, one of the areas of concern is the possibility of unintentional changes. There is a general difficulty in screening for these unforeseen changes since it is a conceptual leap to anticipate the unexpected. However, in general the approach has been to examine general performance of the new cultivars like agronomic performance and compositional analysis to detect unintentional effects. PIP products can be both transformed lines and the result of traditional breeding of two transformed lines to yield a new product with combined traits like WideStrike cotton. In both cases, the new PIP product must be registered just as other new combinations of pesticide active ingredients must be registered.

For PIP products resulting from traditionally bred transformed lines, under what circumstances, if any, would it be appropriate to examine agronomic performance and compositional analysis to provide a screen for unintentional changes in the crop? Please describe other ways EPA might consider screening for potential unintentional changes in a crop. Page 16 of 85 Panel Response Agronomic Performance and Composition Analysis Based on the evidence presented in the safety assessment of the single Bt containing strain and the more recent extensive testing of the stacked Cry1F/ Cry1Ac material, the Panel concluded that there is little to suggest that the stacked variety poses significantly more risk or unanticipated consequences to agronomic performance than do varieties with either the Cry1F or Cry1Ac genes introduced singly. The Panel was unaware of any reason to believe that previously undetected and unanticipated changes would have occurred in the plant breeding beyond the uncertainties normally associated with plant breeding and the original transformation process in parental GM lines.

The food safety of the two individual Cry proteins present in WideStrike has already been established. It is worth noting that cotton fiber is not a food and does not contain residual Cry proteins. Cotton meal, which may contain small amounts of Cry proteins, is usually only fed at low levels to ruminants because they cannot tolerate larger amounts of gossypol which is relatively toxic to non ruminant animals. Cottonseed oil is a bleached and purified product that contains only trace amounts of protein. Cottonseed oil is one of several vegetable oils consumed by humans and thus is a relatively minor dietary component.

Composition, phenotypic traits and agronomic performance can be powerful tools for revealing unintended effects and can be viewed as screening methods since they are the end result of the balanced and coordinated expression of numerous multigenic traits. Plant breeders normally eliminate from further development plants with visible or compositional defects or undesirable alterations. Compositional or agronomic studies are normally not required on plants resulting from traditional breeding.

Suggested Additional Procedures To Screen For Potential Unintentional Changes In the Crop For the Panel's response to the question, the Panel considered the screening for unintended changes to WideStrike and, on a broader perspective, other plant incorporated protectants ( PIP) and genetically modified crops. Screening for unintended effects is encouraged. Such a screening could use profiling methods for unintended effects, e. g. expression microarrays or metabolomic studies ( e. g., as discussed in SAFOTEST ( http:// www. entransfood. nl/ RTDprojects/ SAFOTEST/ safotest. html). However, as these tests are still under development, toxicology studies in rodents, looking for effects of unintended effects, although unlikely, could be appropriate. While the targeted safety assessment system in place today has apparently been effective in risk characterization, it cannot eliminate the chance that unintended or unexpected consequences of plant breeding will escape detection. It would be extremely useful to have comparative data regarding the frequency and magnitude of unintended effects associated with conventional plant breeding with or without the use of genetic engineering techniques upon which a quantitative risk assessment could be based. The Agency could play an Page 17 of 85 active role in advancing comparative risk assessment by supporting research, perhaps in cooperation with FDA and USDA, on unintended effects associated with plant breeding.

Technologies such as metabolic profiling, gene expression systems and proteomics might be useful for evaluating unintended effects. These methods need to be more fully developed and their utility documented before they can be used to inform the scientific review. Previous reports such as The US General Accounting Office ( GAO), in its assessment of the FDA's safety testing of GM foods ( GAO 1992) and several European entities ( http:// www. entransfood. com) have concluded that non targeted profiling methods might be useful in assessing the risks associated with unintended effects. The Panel recommended that the EPA, in partnership with FDA and USDA, support research on the development and validation of profiling methods and provide support for the further development of site specific gene insertion techniques. The Panel's detailed comments are presented below.

Potential unintended effects of GM foods on human health Unintended effects of traditional breeding methods on levels of anti nutritional or toxic constituents in food organisms have been characterized in conventional organisms. Organisms derived from conventional breeding methods including tissue cultures may have a somewhat enhanced possibility for genetic ( and epigenetic) instabilities, such as the activity of mobile elements and gene silencing effects ( Bhat and Srinivasan 2002). These effects could result in an increased possibility of pleiotropic unintended effects, e. g., increased or decreased expression of constituents or possibly modifications in expressed proteins as well as epistasis, ( the interaction of the inserted gene with other genes).

In general, the compositional analysis should be performed on the basis of validated scientific methods. Strategies for the compositional analysis in food products derived from GM plants have been established, where key substances are identified and analyzed per species. Furthermore, in order to be able to interpret the data from the compositional analysis of individual animal products adequately, insight into the natural variation in the relevant macro and micronutrients and antinutrients, if present, will be required.

In the future, compositional analysis may also be based on unbiased profiling of the GM food product and the conventional counterpart. Techniques for the profiling approach are now under development and can be divided into three subsections: genomics, proteomics and metabolomics to screen for differences in the GM plants in relation to the gene transcription products, proteins and metabolites, respectively. At the moment, however, none of these techniques is yet validated and ready for routine use in risk assessment ( WHO/ FAO expert consultation, 2003).

The Molecular Biology of Unintended Effects The problem of assessing unintended effects has been a matter for scientific discussion for a long time and the principles of assessment by international scientific organizations have changed considerably. For example, the use of the concept of the substantial equivalent has been criticised Page 18 of 85 extensively, and the idea of the principle has been reduced to its use as a starting point of a risk assessment.

In principle, traditionally bred transformed lines should have the same characteristics as the parent organisms. However, analyses of events have pointed to potential different outcomes in some cases where the basis of these events is not presently fully understood.

Two specific issues for consideration are:

( 1) A molecular characterization of the new product should show that the recombinant traits/ sequences in the new product are identical to the insertion / traits in their parental lines. In principle, the method of breeding the two parental lines could affect the genetic characteristics of the inserted traits. Therefore, evidence is needed that no such effect has occurred. For these products, a risk assessment combining evidence from parental lines and the product should be requested.

( 2) Stability is another concern. For other similar products, it is uncertain how many generations need to be observed to establish stability of inserted traits.

Sites of Insertion of Foreign Genes and Potential Effects One of the concerns with any genetically modified organism is the precise location point( s) of the inserted gene or genes. Single copy insertions of the genes at locations other than in functional gene regions or in regulatory areas is the goal but with current technologies insertion is typically relatively random. If significant anomalies occur they will surely be picked out and discarded through breeding programs but more subtle effects might possibly slip through. In the specific case at hand, the utilization of Cry1F and Cry1Ac genes incorporated into the cotton genome, it is noteworthy that both for the single insertions via typical recombinant DNA techniques and then the construction of the Cry1F/ CRY1Ac stacked strain by traditional breeding involved only single insertion events in each case.

Potential health or environmental risks of genetically combining T DNA transgenes in WideStrike cotton through traditional breeding are unlikely to differ from those of the parental transgenic lines. However, our present knowledge concerning unique or special risks associated with GM crops is somewhat limited and presently under study ( NAS, 2002). One ongoing concern is that T DNA insertion into the genome is a mutagenic event, which could trigger unintended changes in nutritional or toxicological properties of a transformed crop ( Schubert, 2002). T DNA insertions that disrupt agronomic performance or gross chemical composition can be detected by current methods of screening, whereas other insertions that alter biosynthesis of molecules which are toxic, allergenic or carcinogenic would likely be overlooked. Given that there are no a priori means to predict these different outcomes, additional information should be sought on potential genetic effects of T DNA insertion, as outlined below.

T DNA inserts and effect on genome organization Page 19 of 85 Assessments of risks posed by GM crops rely on comparisons with conventionally bred ( non transformed) counterparts having a history of safe use. Apart from comparing agricultural characteristics and chemical composition, GM plants are also routinely analyzed for the number of T DNA insertion sites, the number of gene copies at these sites, and the organization of DNA within inserts. In addition, the effect of T DNA on proximal open reading frames of cellular proteins is also considered. However, additional measures outlined below would better inform us of potential downstream genetic effects associated with transgenic insertion of foreign genes.

For instance, routine Southern hybridization analysis of WideStrike cotton and its parental transformed lines suggest that T DNA inserts coding for individual insecticidal proteins are present as single copies in the cotton genome. The importance of this observation is that single transgene inserts breed true and show fewer unwanted effects ( i. e., transgene silencing or mutations) than multi copy transgene lines. However, single copy T DNA inserts are known to trigger large scale chromosomal rearrangements, including translocations ( Forsbach et al, 2003). In such cases, large portions of flanking DNA at the T DNA insert site may also be duplicated and translocated to multiple chromosome locations ( Nacry et al, 1998, Tax and Vernon, 2001). These types of chromosomal rearrangements would likely complicate interpretations of Southern hybridization data, by suggesting that a single copy of the transgene was present, when in fact duplication and translocation of the T DNA resulted in an additional chromosomal copy with identical flanking sequences. Therefore to complement and extend current strategies for assigning transgene copy number, the chromosome location of each T DNA insert should be routinely mapped through use of Mendelian segregation results or molecular markers for cotton ( Tomkins et al, 2001; www. genome. clemson. edu/ projects/ cotton/ bac and www. cottoninc. com/ Agriculture/ homepage. cfm? page= 3157).

A related concern is whether T DNA induced mutations are qualitatively or quantitatively similar to those which occur as a consequence of traditional crop breeding. It is known that crossbreeding of genetically different cultivars can result in major chromosomal rearrangements. In addition, mobile DNA elements ( transposons) also induce genetic changes in plants. At present, it is far from clear whether plant transformation differs from these processes and poses unique or greater genetic risks.

T DNA inserts and effect on gene expression Several hundred base pairs of genomic sequence were identified by sequencing of regions flanking T DNAs bearing the Cry1Ac and Cry1F transgenes in WideStrike and parental cotton lines. When used as in silico probes, BLAST searches failed to reveal significant homology between these sequences and those previously deposited in the GenBank database. Moreover, analysis of 1032 bp ( 534 bp from 5' border and deleted 16 bp + 482 bp from the 3' border) of cotton sequences flanking the Bt Cry1Ac cotton 3006 210 23 insertion site revealed no significant (< 450 bp, 150 aa) open reading frames at this cloned locus. Similarly, analysis of 5028 bp ( 2073 bp from 5' border and deleted 53 bp + 2902 bp from the 3' border) of cotton sequences flanking the Bt Cry1F cotton 281 24 236 insertion site revealed no significant (> 450 bp, 150 aa) open Page 20 of 85 reading frames at this cloned locus. Although cotton genes were thus not computationally identified at the T DNA insertion sites, it is unclear why attempts were not made to determine whether these flanking sequences correspond to one or more transcriptionally expressed cellular genes.

In this regard, it is noteworthy to consider that T DNA is widely used by plant biologists as a mutagenic agent to study the biological roles of specific genes ( Forsbach, 2003). Moreover, T DNAs in the present application contain strong agrobacterial enhancer sequences ( 4ocs) which are known to drive expression of genes that are proximal to the insertion site [ this process, known as " activation tagging" ( Tani H, 2004), is widely used in modern plant biology research to identify novel genes.] Thus to more fully investigate the possibility that T DNA inserts are in active genes, Northern blot hybridization to detect cognate mRNA transcripts should be done using 5' and 3' sequences that flank each T DNA as probes. Comparing the size of transcripts detected by Northern blot in fractionated RNA from nontransformed and transformed lines will assess whether the T DNA insertion physically disrupted or significantly ( 2 SD from the norm) affected expression of the cellular gene. It is also recommended that due to potential " enhancer effects" by multiple ocs promoter sequences, additional flanking sequences 5' and 3' from the T DNA should be identified to characterize genes that are distal to the insert site. Expression of these distal genes would be monitored by Northern blot analysis or through more systematic investigations using DNA microarrays.

Unintended effects that may result from the insertion of DNA into the plant genome represent hypothetical hazards that, as noted previously, have yet to be demonstrated to occur in products presented to the Agency for review. The challenge is to define specific biochemical or metabolic changes that represent risks that warrant rejection or management. This requires specific evidence of a molecular or compositional change that would trigger further investigation.

It is impossible to quantify and characterize the risk associated with unintended effects without defining it in terms of molecular or metabolic changes. It bears repeating that while unintended changes may be possible, it is both not clear that they present any new or different risks that are significant ( meaningful to human and animal health). To date no evidence has been presented that significant risks occur ( Beachy, 2002, Kuiper 2001).

Conventional plant breeding itself produces unintended effects. There is a long history of safety associated with plant breeding. Historically, plant breeders have depended on phenotype and performance to detect unintended effects and reject plants harboring undesired characteristics. In some cases, compositional studies may inform the selection of individuals worthy of further development. One is reminded of the fact that plant breeders have successfully employed strong mutagenic techniques such as chemical mutagens, UV light, and radiation to produce genetic diversity and altered traits.

New Possibilities for Gene Insertion Major advances in techniques for precise placement of foreign genes into host organisms Page 21 of 85 should very soon render these particular concerns about unintended consequences of foreign gene insertion relatively insignificant. Recent research has shown that highly precise implanting of foreign genes into specific sites in a host genome is not only possible, but can be accomplished fairly readily. The work of Lambowitz and his colleagues at the Institute for Cellular and Molecular Biology at the University of Texas at Austin on site specific DNA insertion through the use of autocatalytic group II introns is notable in this regard ( Karlberg et al., 2001, Mohr and Lambowitz, 2003). While such positional gene insertion will certainly serve to greatly ameliorate unintended effects, it will not eliminate them entirely, As Schubert ( 2001) and others have pointed out, genes can act at a distance in unexpected ways. New compounds or pathways might be created or activated and unexpected associations between molecules may occur.

Animal Feeding Studies for Unintended Effects It is often suggested that animal studies should be used as a non targeted screen for undetected changes for PIPs. There are several limitations to the use of animal studies on whole foods. The first challenge is that it is often difficult to formulate a diet that is nutritionally adequate and well accepted by the test animal which also contains a large percentage of a whole food ( FAO/ WHO, 2000; Chassy, 2004). Animal studies have proven useful for the evaluation of pharmaceuticals, food additives, industrial and agricultural chemicals, and environmental contaminants because these compounds have biological activity at fairly low levels of exposure. Carefully performed toxicology studies employing various concentrations of pure chemicals can be used to determine a lowest observed adverse effect level ( LOAEL) in a particular animal species, typically a 90 day rodent study. Such studies allow the setting of maximum permissible exposure levels that provide an acceptable safety margin. It is often impossible to devise an animal diet with a whole food such that it contains a sufficient quantity of a toxicant that will elicit a biological response. Chassy et al. ( 2004) indicated:

" A review ( Munro and others, 1996b) of 120 rat bioassays ( each of 90 day duration) of chemicals of diverse structure including food additives, pesticides, and industrial chemicals found LOAELs to range from 0.2 to 5000 mg/ kg body weight with a median of 100 mg/ kg and a 5th percentile of 2 mg/ kg. To achieve the 5th percentile of exposure from a toxic constituent present in, say, a food crop in a rodent bioassay ( at a food incorporation rate of 30%) the toxin would have to be present at a level of 80 ppm. To achieve the median exposure of 100 mg/ kg it would have to be present at 5000 ppm. These concentrations fall well within the range of existing analytical techniques for detection of inherent toxicants in food. The concentrations should also be readily detected during compositional analysis of the known toxicants in the host organism used to generate the improved nutrition crop."

It is clear that analytical techniques have far greater power than animal studies for the detection of individual compounds. Moreover, animal studies on whole foods suffer from the lack of a specific targeted hypothesis as well as the possible presence of a variety of confounders. It is much easier to design a study to investigate an effect on a specific target organ, enzyme level or serum metabolite concentration than it is to compare the health outcomes of two diets. Although several whole food studies have been reported ( Chassy 2004), it may be more appropriate to use Page 22 of 85 animal studies to probe for suspected metabolic or toxic effects on a case by case basis than it would be to require them in every case. It is also not clear that whole food animal studies would extrapolate very well to human populations that have great genetic and dietary diversity. Animal studies are therefore not likely to be an effective screen for unintended effects.

Targeted Compositional Analysis Phenotype and functionality are highly complex traits. Expression of literally thousands of genes must be coordinated in both extent and timing. Expression is also modulated by extrinsic environmental signals, availability of nutrients, pests, diseases and a host of other factors. Phenotype and function are therefore highly sensitive indicators that should not be regarded lightly.

Compositional studies are also powerful indicators that no significant unintended effects have occurred in the breeding and development process. There are three important points to make about composition. The first is that " we are what we eat." It follows that all we need to know is the safety of the components of the food we eat. The only thing that really matters in a safety assessment of a food or ingredient derived from a transgenic crop is the composition of the product that consumers will eat. Levels of transcription and translation as well as varying rates of turnover of protein and mRNA normally occur in all organisms. If these natural oscillations do not result in compositional changes that have health or safety implications, such changes are of little consequence.

The second important point to be made about composition that is often misunderstood is that there is a natural range in concentrations observed for many metabolites in a plant. It is misleading to look in a food composition database and conclude that maize is composed of 9.5% protein. The number in the database represents an average value that may be reasonably used for calculations of, for example, protein content of a diet. It does not, however, reveal the wide range of concentration of proteins ( and all other metabolites) that may be found in individual samples of maize. It is not possible to draw conclusions about unintended effects related to changes in composition without understanding the natural variability in natural product composition. Until recently, very little information was available about natural variability in composition. ILSI has recently placed a free and easily accessible crop composition database online ( http:// www. cropcomposition. org/). The utility of the database has been described in a recent publication ( Ridley et al., 2004).

In many instances, the content of secondary metabolites that are of interest for their nutritional, health beneficial or health protective effects may be even more variable than macronutrients. A health claim has recently been approved for soy protein consumption by the FDA. Many consumers are interested in increasing the soy and isoflavone contents of their diets in response to reports of various potential health benefits. The data presented in Table 1 demonstrate that soybeans vary greatly in isoflavone content. The same cultivar grown at 4 different sites gave rise to a nearly 4 fold difference in isoflavone content. Almost 3 fold differences were observed between four commercial varieties of soybean. Page 23 of 85 The third point to be made about the value of composition studies is that they are powerful sensing probes for the concentration of almost all the remaining cellular metabolites. This is because many of the analytes that are evaluated in composition studies ( i. e., amino acids, fatty acids, vitamins, toxicants such as gossypol, etc.) are products or intermediates in one or more metabolic pathways. Cell metabolism is a highly integrated and interconnected set of highly regulated enzymatically catalyzed pathways. Knowledge that the concentration of dozens if not hundreds of metabolites is within normal range is strong evidence that most all metabolites are within normal ranges. It is therefore not necessary to measure each individual metabolite. Perhaps more importantly, the currently used targeted composition studies often account for over 99% of the total composition and it specifically targets micro components that are of health or safety significance ( i. e. toxicants, vitamins, bioactive phytochemicals, allergens, antinutrients, etc.). It is worth repeating that there is no scientific evidence that targeted composition studies that evaluate over 100 analytes from diverse metabolic pathways have failed to detect significant risks. Moreover, there is also no evidence that the kinds of hypothetical unintended effects that might arise from gene insertion do not also occur in conventional breeding. Several unintended effects of this kind have been reported to occur as a result of conventional breeding programs ( Kuiper et al., 2001).

It is important to stress that not only does content of specific metabolites vary over a broad range in plants, but also that changes in the composition of a plant may be beneficial. In fact, numerous development projects are in progress that seek to alter the content of macro or micronutrients, eliminate allergens or toxicants, or introduce other compositional changes that may be health beneficial. Golden Rice is a well known example of this kind of strategy ( Potrykus 2001). A new risk assessment paradigm may need to be created in order to assess the unintended changes that might accompany large changes in composition.

Almost all of the first wave of biotechnology derived crops that have been approved and commercialized were designed to be no different than conventional varieties of that crop except for the addition of one or two additional traits   often intended to enhance the agronomic properties. While the Panel indicated that GMO crops were considered as safe since being shown to be " substantially equivalent" to their conventional counterparts, but this is a misunderstanding of the concept of substantial equivalence ( FAO/ WHO, 2000). Substantial equivalence uses compositional analysis to identify differences that merit further risk characterization so it is in effect a comparative assessment paradigm. The finding of differences per se, does not demonstrate an increased risk. As a consequence of misunderstanding of the substantial equivalence paradigm, it has been suggested that it should be evolved into the Comparative Assessment paradigm ( Kok and Kuiper 2003). The use of the comparative assessment concept also lends itself to safety assessment of compositionally modified novel foods ( Chassy, 2004). The Agency can reasonably expect to be asked to review crop plants that have PIP( s) and nutritional enhancements so it is essentially to accept that composition difference is now and will increasingly be in the future a fact.

It should also be noted that human diets vary by an even greater range than the Page 24 of 85 composition of the individual components in the diet. This has enormous consequences for public health. The comparative safety of any unintended changes must always be related to exposure in the context of the whole diet and the range of dietary intakes represented in the particular subject population ( Chassy et al., 2004).

Metabolome An excellent recent review by Goodacre ( 2004) described the current state of art of technology and understanding of metabolism and metabolomics.

The Panel commented that translation ( proteome) and transcription ( transcriptome) may provide useful insights into cell function and metabolism, and the composition of the food derived from a plant that determines fitness for human or animal consumption. It is instructive to investigate methods that might shed additional insight into what has now become referred to as the metabolome. One approach would be to conduct a comprehensive analysis of the concentration of each cellular metabolite. One could envision a rapid parallel analyzer that would provide a complete blueprint of the metabolome. The short coming of this approach is that databases that establish norms for each metabolite would need to be constructed, the normally observed ranges in concentration documented, and concentrations of each metabolite that has safety implications determined. There is at present little data that would relate nutrition, development, performance and health of animals to more than a few dozen compounds, primarily the nutrients. The development of non targeted metabolic screening methodology as well as paradigms for understanding the implications of specific concentrations is a subject of research. We may be some years away from this kind of metabolic profiling, although in the near term some metabolite screening technologies may prove useful in identifying changes that could then be evaluated by more targeted methods in order to understand their significance.

In the meanwhile, there is another approach to metabolome screening that might be called metabolic fingerprinting. It is a logical extension of the composition analysis that is now used to assess the safety of transgenic crops. It should be possible to look at the metabolic pathways in a plant cell and select a dozen or two dozen key metabolic intermediates that strongly correlate with overall cell composition. The information required to do this is in the literature and expert physiologists and biochemists could probably reach a consensus on which molecules to select for studies aimed at validating this intelligent targeted fingerprinting.

There is, however, no way that such studies can cope with totally novel metabolites that arise from newly formed pathways or activation of silent genes. This would be in effect, " knowing the unknowable." Fortunately, the multi pronged safety assessment process employs several distinct types of tests ( as noted above) in order to screen for significant unintended effects that would be undetected by compositional analysis.

Metabolic Profiling Plants produce an extraordinary array of small compounds and these can affect all stages Page 25 of 85 of development as well as the properties of the final materials from these plants that are used in commerce. In the past, analysis of such metabolites has essentially been on a case by case basis and has been limited to only a few chemical entities. Nutritional and compositional assays of GM plants examine such properties as the levels of proteins, sugars, fats and other materials. These targeted assays are certainly extremely useful. However, when screening for potential unanticipated consequences of foreign gene introduction, it would be very desirable to have very broad yet precise indicators of what is going on in the modified and non modified plants as far as metabolites are concerned. New tools for looking at issues such as metabolic profiles are coming into widespread use and these could provide a much more broad band examination of modified and non modified plant materials in a cost effective manner.

The recent techniques that have become available can detect and quantify several hundreds of plant metabolites in a single analysis ( thus the term metabolic profiling), using techniques such as gas chromatography/ mass spectrometry ( GC/ MS) and liquid chromatography/ mass spectrometry ( LC/ MS). Such procedures lend themselves well to providing a more detailed understanding of the potential effects of foreign gene introduction into plants. From the evidence available to date, it is very likely that few differences will be seen between the already heavily studied GM crops and their non GM counterparts. It would be very reassuring that at a basic level, little metabolic disturbance was occurring because of the transgenic alterations. Finally, testing of new genetically altered materials in this manner, early on in the development process, would serve to provide an early warning of additional concern.

Proteomics Future studies of genetically engineered plants using proteomic approaches look very favorable for comparison of transformed and non transformed varieties as well as for traditionally bred variants of transformed species. As has been expressed by Voet and Voet ( 2004) proteomics investigates " all proteins expressed by an organism, with an emphasis on their quantification, localization, modifications, interactions and activities. " Tools used to gather such information include 2 D gel electrophoresis combined with mass spectrometry, amino acid composition and sequence analysis, peptide mass fingerprinting and matrix assisted laser desorption ionization ( MALDI) time of flight ( TOF) mass spectrometry for identification of post translational modifications. While these processes can be highly precise and definitive in ascertaining differences between samples, for full utilization they do depend upon the availability of adequate databases, which are still being assembled for many plant species.

Gene Microarrays The use of gene microarrays to assess, for example, which genes are being transcribed into messenger RNA at a particular time, in a particular organ, tissue type or even sub cellular organelle has been transforming molecular biology in recent years. This same technology can potentially be employed to answer questions about how plant metabolism might be altered by the incorporation of foreign genes, for example those of the Bt toxins. Of course the use of such microarrays in a meaningful manner requires knowledge about the genome of the plant in Page 26 of 85 question. In this connection, it is encouraging to note that sequencing of the cotton genome is proceeding apace, that approximately 50,000 EST sequences are available and that microarrays covering about 30% of the cotton genome are already available ( http:// cottongenomecenter. ucdavis. edu/ microarrays. asp).

These newer techniques of metabolic profiling, proteomics and gene chips are indeed in their infancy as far as application to plant systems is concerned and clearly need database collection, validation, etc. before they are put into regulatory use. Additionally, when this is accomplished, they should be used as adjuncts to present test methodologies, which have served well. What these new tools, with their broad span analysis do offer is great potential in the future for allowing recognition of unintended consequences of genetic manipulation early in the process. This is true whether the genetic changes are made by traditional or recombinant methodologies.

Agency Charge Ecological Risk 1. WideStrike cotton is a product expressing pyramided Cry1F and Cry1Ac Bt proteins. The submitted non target effects studies examined the effects of the Cry1F and Cry1Ac proteins separately and in combination to detect any synergistic effects on non target wildlife. No synergistic effects or increase in non target host range were seen as a result of combining these two proteins in the same product.

The Panel is requested to comment on the need for non target hazard data development on the combinations of Cry proteins being considered for registration when data on the effects of the individual Cry proteins are readily available and show no adverse effects.

Panel Response The Agency has done a good job of preparing documents presenting the available data regarding control of TBW, CBW and PBW in cotton. Cry1Ac and Cry1F seem to be rather specific. Thus direct hazards to vertebrates due to exposure to the Cry1F and Cry1Ac proteins should be minimal or non existent, making consideration of synergy in vertebrates unwarranted. Hazards to invertebrates have a higher likelihood of showing effect. In fact, synergy has been observed for Bt toxins and spores in various lepidoptera ( Moar et al. 1989, Dubois and Dean 1995, Johnson and McGaughey 1996), and between different CryIV and CytA toxins in Aedes aegypti ( Chilcutt and Ellars 1988). In contrast, other studies have failed to demonstrate any synergistic interactions between Cry1A toxins in various lepidopteran insects ( Moar et al. 2002, by Tabashnik 1992). One citation ( Chakrabarti et al. 1998) indicated a synergistic interaction between Cry1Ac and Cry1F in Helicoverpa armigera. Access to the article would have assisted the Panel to fully examine the experimental methods and analytical techniques. Although interactions between toxins and spores would not be an issue with transgenic plants that express only the toxins, potential interactions among different Cry toxins may be important in resistance management ( e. g., Tabashnik et al. 1997). Considering all data presented and information from Page 27 of 85 one literature review, the predominance of data demonstrated no synergy for Cry1 proteins. Therefore, the Panel concluded that synergistic effects are unlikely to occur when plants produce Cry1Ac and Cry1F at the concentrations that are currently needed to control TBW, CBW and PBW.

When each toxin was tested independently and in combination at environmentally relevant concentrations, none of the data indicated any synergistic or additive effects on the target or nontarget organisms tested. Evaluations of non target organisms were generally done at rates far in excess of what these organisms would be exposed to in the field.

Based on the written and verbal information provided by the Agency, the Panel believed that non target hazard data can be treated similarly to those data in the Human Health Risk Assessment, for which a similar question, as noted previously was raised. Indeed, this is a very similar issue, since mammals are also non target organisms. Therefore, the Panel concurs with the opinions the Panel expressed in the answer to Question 1 from the Product Characterization/ Human Health Risk Assessment, that toxicity testing on the combinations of Cry proteins is not necessary. The Panel's opinion that Cry proteins do not require further synergistic testing will not necessarily extend to other types of Bt toxins.

While synergism is not considered to be important for the Cry1 proteins under consideration, the Panel believed that future testing of non target effects of these types of toxins should proceed, with both toxins to be expressed by genetically engineered plants rather than with individual purified toxins. Testing mixtures of compounds would provide data that are more relevant to the proposed use scenarios and likely exposure scenarios. Testing the toxicity of mixtures for several non target insect species would also provide data that could refine field evaluations to determine any effects on insect species diversity. This could be a critical parameter controlling food resources for, and thus impacts on avian and mammalian species. Such an evaluation will ultimately provide data that could be used in ecological risk assessments where the question that should be asked is: What is the combined effect of Cyr1Ac and Cry1F on species elimination from ecosystems?

Some concern was raised regarding the small amount of Cry1F data found through a CAB search of the literature since 1973. A single non target study of monarch butterflies ( Helmich et al. 2001) and 11 studies involving target species were found. The Panel suggested: a) USDA has compiled more Cry1F data than is available in the materials prepared for this review or in the peer reviewed literature; b) it would be useful if all non target data from the Agency presentation, the USDA evaluations, and peer reviewed literature be compiled into one summary table or a series of tables for reference purposes in future evaluations of this and similar products.

Agency Charge 2. The weight of evidence from the reviewed data indicates that there will not be a hazard to wildlife from the commercialization of WideStrike cotton. Although the Bt proteins expressed by WideStrike are known to affect only lepidopteran insect species, the Agency Page 28 of 85 evaluated studies of potential effects on a wide variety of non target organisms that might be exposed to the Cry1F and Cry1Ac protein, i. e., wild mammals, birds, invertebrates, and aquatic species. EPA concluded that aquatic and terrestrial wildlife was not likely to be harmed and that WideStrike cotton was not likely to threaten the long term survival of any non target wildlife populations.

The Panel is requested to comment on the Agency's analysis of the currently available data on the potential impacts of WideStrike cotton on non target species.

Panel Response The Panel recognized improvements in the quality of the Agency's analysis and the degree to which this reflected a positive response by the Agency to recommendations made by previous SAP reviews of PIPs. The Panel also noted that the Tier I tests were generally of a higher quality than the tests that other SAPs have reviewed. Overall, however, detailed analysis of the fieldbased evaluations revealed that these did not meet current standards, and the Panel repeated requests made by other SAPs, that the Agency issue detailed guidelines for semi field, and fieldbased procedures. The Panel recommended that the exposure pathway and diet used within the Chrysoperla test be examined further to determine the rigor and repeatability of the test and also suggested that Orius spp. be considered as a more appropriate test subject. The Panel also considered that a more appropriate parasitoid than Nasonia should be selected. Finally, the Panel expanded the table of suggested arthropod test organisms, first developed by the August 2002 SAP, to include aquatic species. A detailed response to the question by the Panel is provided below.

The Panel noted improvements in the quality of the Agency analysis and assessment since the Panel last met on this subject approximately two years ago. Given that some of these improvements appear to have been in direct response to recommendations made by previous SAPs, the current Panel argued that this highlighted the value of the public review process and that it also provided evidence of a positive response to SAP comments by the Agency.

With respect to the Tier I screening procedures reported for WideStrike cotton, the general quality of the submissions was considered to be better than in previous cases, particularly with regard to characterization of test material in the bioassay procedures, the selection of test organisms and the quality and clarity of the reporting. The Agency review was found to be more informative of its logic and reasoning, and more explicit about the limitations of the tests. This made it easier for the Panel to be constructively critical.

The Panel had general comments concerning maximum hazard dose evaluation. The Panel did not find the protocols for Tier 1 testing appropriate. The duration of many tests were determined by control mortality exceeding 20%. The Panel suggested that control mortalities as high as 20% raise a concern of an error with the protocol and that tests either need to be repeated or that some adjustments in protocols are required. Sample sizes also seem small, providing little power to discern differences that may exist. Page 29 of 85 The greatest challenge raised by the Panel mainly arose through the lack of progress with development of EPA approved protocols for field based evaluations, clarity regarding their role relative to laboratory based evaluations, and the availability of intermediate options that might mitigate the need for long term, open ended, field studies, that are not designed to test clearly stated hypotheses. The Agency acknowledged that specific protocols are lacking and that science needs to advance in order for protocols to be developed. Thus, field data are treated as supplementary by the EPA, and procedures are not subjected to the critical scrutiny that is the norm for required tests.

With regard to the maximum hazard approach, the effectiveness of this approach is dependent upon species selection ( where a range of relevant physiologies should be evaluated, relative to the mode of action of the test material), and rigor in the conduct of tests, which should follow detailed protocols. The Panel noted that the tests reported meet many of the criteria listed by previous Panels, but, it argued that in order to affect permanent advances in the standards of testing, criteria for the conduct of tests need to be built into new written requirements or test guidelines.

Of particular importance for inclusion in these new guidelines are criteria noted by two previous SAPs ( in 1999 and Aug 2002). These include: 1) verification of exposure levels of test organisms to proteins throughout the bioassay; 2) detailed quantification of EEC in the field; 3) clearly stated endpoints; 4) a clear statement that tests which fail to reach the designated endpoint are not eligible for consideration; 5) use, where possible, of foods used by test species in their relevant habitat; 6) verification that the food offered to the species actually contained the administered material, at the intended dose, throughout the investigation; 7) verification that all life stages of the species are exposed appropriately to the transgene product ( i. e. actually contact the toxin in relevant ways); and 8) that there is sufficient replication and that sufficient numbers of insects were screened based on statistical power.

The Agency stated that field testing was " recommended by the August 2002 SAP". Ultimately, scientific confidence in Tier I screening should be sufficient to limit further tiers of testing when no effects are detected at the maximum hazard dose. This would mean that no open field investigations would be undertaken with some products. Confidence in this approach would increase if:

  The armory of guidelines were to be expanded to include extended laboratory and semifield approaches as an intermediate step, before full field testing was requested. No effects or failure to exceed a trigger or threshold value in these tests would cause the testing to cease at that point. Widely used intermediate testing methods include extended laboratory tests ( use of more realistic substrates and exposure pathways within the laboratory) or semi field tests ( confinement of individual or multiple species of test organisms within microcosms, mesocosms, field cages or barriered arenas). The Agency is encouraged to avail itself of opportunities to discuss method development in the international arena, for example in relevant working groups of such organizations as the International Standards Page 30 of 85 Organization, OECD, International Organization of Biological Control, and SETAC, among others.

  Bioassessment protocols were developed for surveys in whole fields to help answer concerns about the possible detection of rare events, effects resulting from combinations of treatments that do not occur within conventional experimental designs or concerns about long term, unforeseen effects. Such protocols could then be employed on the health of agroecosystems, or as a component of post release product stewardship.

  The field evaluation guidelines presented were greatly improved with respect to experimental design, sampling procedures, taxonomic focus, statistics and interpretation.

The Panel reiterated its recommendations from the August 2002 FIFRA SAP meeting regarding:

  The requirement that registrants evaluate and select test sites from a number of candidates, in the season before testing begins, to determine whether the organisms of concern are present and sufficiently abundant to provide a basis for statistical discrimination of small but significant effects.

  Use of sampling methods of known efficiency and precision with consideration to within plot variability when determining the intensity and frequency of sampling.

  Use of a scale and layout of the experiment that minimizes the risks of edge effects and reinvasion from untreated control plots, and which takes into account the dispersal rates and phenology of the organisms of concern.

The Panel recognized the limitations of laboratory based testing, and it may be argued that the requirement for field testing will continue until a dataset has been generated that effectively validates the maximum hazard method. The most important limitations to laboratory testing, that can be compensated for by field tests, were noted by the August 2002 FIFRA SAP and included the statements that:

  Levels and routes of test material exposure that may not be realistic in the laboratory.

  Effects assessments are made after short term exposure of organisms, not lifetime exposures as might occur in the field.

  Organisms in the field are subject to supplementary stresses that have additive effects, including sub optimal temperatures and humidity, and starvation and parasitism, that amplify impacts that occur under the optimal physical and biological conditions of laboratory tests. Page 31 of 85   Laboratory tests may evaluate an appropriate category of organism, but they inevitably fail to evaluate species that are actually exposed to test substances in the field.

Despite these limitations, the Panel argued that the Agency could already be striving for a more structured approach to risk assessment for PIPs, guided by more detailed protocols, that expand the availability of replicated intermediate field test data, which increase the potential for long term, multi field surveys after release, and which probably decrease the expectation for open ended field census studies with their various methodological challenges ( as noted in the August 2002 meeting minutes).

With respect to the avian hazard assessment, the Panel was asked to consider the effects of a reduced food base for birds utilizing corn fields that have a high non target insect mortality. Are nestlings any more susceptible to Cry proteins after they have been biotransformed by the insect and fed to the nestling? Do birds lack the bioactivating enzyme or the actual receptor site for the toxin? Could songbird or game bird nesting success change with WideStrike cultivation relative to conventional cotton cultivation? Perhaps insect prey availability would be sufficiently different to produce a difference in nesting success. This question emerged from UK work with grey partridge and other species impacted indirectly by agrochemicals.

With respect to aquatic species, the Panel noted that it was correctly stated by the Agency that the August 2002 SAP recommended a list consisting only of terrestrial taxa for Tier 1 testing. The Panel noted however, that this does not mean that aquatic taxa were excluded as being potentially affected in some circumstances, even if not in the present case for cotton. A proposal was made to extend the table of proposed test organisms in the 2002 FIFRA SAP meeting minutes to indicate where aquatic taxa may be incorporated. It was also suggested from the August 2002 FIFRA SAP meeting minutes that Daphnia might be substituted by a member of the shredder functional feeding group ( e. g., some Diptera or Trichoptera species might be more appropriate) to increase the opportunity for uptake of the toxin. Precipitate formation in the reported Daphnia test was difficult to interpret, but may imply that the test organisms were not exposed to the test substance. Page 32 of 85 The table of recommended test taxa from the August 2002 FIFRA SAP meeting minutes for selection of test taxa has been edited as follows ( underlined):

Functional Group Examples Anthropocentric Functions Secondary pests  Sporadic pests, induced pests Natural enemies  Predators, parasitoids, parasites, competitors, ants, and weed eating herbivores Rare or endangered species  Red list species or species of value for biodiversity conservation Species that generate income  Honey bees, silk moths Species of social or cultural value  e. g. Monarch butterflies, honey bees, organisms conferring health of lotic or lentic aquatic systems associated with crop or near crop habitats Ecological Functional Groups Non target herbivores  Plant eating species that are not the target of the transgene ( including aquatic species if appropriate) Secondary consumers  Species that eat herbivores; predators, parasitoids, parasites Pollinators  Bees, selected Diptera ( e. g. Syrphidae) and Coleoptera, etc. Decomposers  Scavengers, ants, Collembola, micro organisms, earthworms, mites, nematodes, and aquatic taxa, particularly those within the shredder functional feeding group Seed dispersers  Birds, small mammals, ants Concerning possible long term impacts on soil invertebrates, the hazardous outcome of impacts on soil taxa was narrowly defined as crop residue build up in the field. This should be expanded to acknowledge the need to protect soil health and ecological function. Residue build up may be one unintended negative consequence of impact on soil organisms that could reveal more significant harm to biogeochemical processes.

Previous Panels have recommended changes to the list of non target insects to be tested and those same concerns are raised again here. Nasonia is a poor model as a parasitoid in the cotton system and there would be many other more suitable models ( Cotesia, Trichogramma, etc.). Chrysoperla is a good model for the cotton system, but others are just as good and maybe better from the standpoint of greater exposure to toxins via plant feeding. Orius insidiosus has been suggested in past Panels and would be a good model because it is very common and abundant in cotton and also feeds on pollen and plant sap. ( The Panel noted that the minute pirate bug is O. tristicolor not insidiosus). Geocoris punctipes would be another very good model for the cotton system. This species occurs in high abundance throughout the cotton belt. It is also a Page 33 of 85 well known plant sap feeder and feeds on small caterpillar larvae. Both Orius and Geocoris are available commercially and easy to rear in the laboratory.

The registrant was congratulated for addressing the issue of toxin decay in the presented diets via ELISA and SDS PAGE. However, the Panel commented that with a small amount of additional testing, the detection of toxins in the non targets themselves would be possible and prudent.

Concerns were raised by the August 2002 FIFRA SAP on Bt corn relative to diet exposure in Chrysoperla. It is not clear that this issue has been satisfactorily addressed here. The toxin is still present only on the surface of the moth eggs. Are the insects ingesting the toxin? Could this be tested by ELISA? Is the registrant confident that the other insect models tested are actually ingesting the toxins? Could this be verified with ELISA? Previous panels have suggested artificial diets for testing purposes. This would seem to alleviate many of the problems associated with definitive exposure. Artificial diets have been developed for Chrysoperla, Orius and Geocoris. Could these be explored as better models for toxin delivery? One Panel member suggested that while this may be a useful method for exposure, some care needs to be taken. Many diets contain enzymes, pro oxidants and other substances that may affect incorporated toxins. The high level of nutrition may also mask more subtle effects of the toxin or may even enhance their effects. The Panel member suggested that special precautions may be needed to avoid these issues. Several companies hold ARS patents for these diets including Buena Biosystems and Beneficial Insectary and would likely be able to supply the diets. Even if these diets don't permit insect reproduction they may be sufficient for shorter term longevity/ survival studies.

The Panel noted that the Hymenopteran parasitoid test was incorrectly described as a larval test in the Agency's review. It also noted enhanced toxicity in the treatment with both proteins at the maximum hazard dose.

The post emergence mortality in all bee treatments detracted slightly from the confidence one has in the honey bee test conclusions. More confidence would have been warranted if postemergence mortality of controls had not indicated that something other than the dosed toxins was influencing bee viability.

Regarding non target organism susceptibility in general, one Panel member considered an analysis for Cry 1f and/ or Cry 1Ac receptors in non target organisms. Perhaps the standard nontarget representatives could be characterized for presence of Cry1F and Cry1Ac receptors? The scientific literature indicated that lepidopterans are the ONLY group of organisms that are capable of being affected by Bt toxins. However, the Panel member had seen no publications which actually attempted to detect Bt receptors in the spectrum of representative non target organisms   other than mammals. Physical confirmation of receptor absence in non target gut epithelial cells would reduce the level of uncertainty and reinforce the indication that Cry proteins, alone and in combination, exhibit little to no toxicity. In addition, the absence of receptors would negate any Page 34 of 85 concerns for food chain transfer of bioactivated Bt toxins by lepidopterans which may be taken as a food source.

With regard to the field studies that were submitted, the Panel noted EUP restrictions in the first year that limit the area that can be planted. Thus, a series of concerns arise that could reasonably have been considered by the Agency and the registrant in planning, execution, analysis and interpretation of these investigations. Although guidelines are not available, the Panel expected standards of current science to be met.

The Panel noted that page 28 of the registrant review stated that plot sizes in these studies were 1,000 square meters. In fact the scale was far less than this, namely 2,667 square feet in the Arizona experiment and 6,400 square feet in the Louisiana experiment, which were only 50 feet and 40 feet long respectively, with mustard and pig weed, or bare earth barriers to separate plots. Invertebrate sampling was undertaken from the central 40 feet and 60 feet, respectively, in each study, but this still means that samples in neighboring plots include points that are within a few feet of each other. The consequences of this are that predatory invertebrates traverse plots to find prey within the scale of the whole experiment. If these animals survive pesticide or PIP treatments, then the pattern of invertebrate abundance across the study reflects treatments to a degree, but also reflects this redistribution of animals between plots. The data therefore contribute very little towards answering the study objectives, which require treatments to be independent of each other.

The Panel noted that if toxic chemicals are used in a non Bt treatment, organisms may move between plots and succumb to these chemicals within the period of toxicity, if they are susceptible. Reinvasion from neighboring plots then occurs and, once toxicity has declined, effects that would persist on an agriculturally relevant scale are obliterated. These scale and reinvasion effects reduce abundance across the study as a whole and limit radically the ability to discriminate between treatments. It is impossible to determine from small scale field tests, how toxic pesticides are to beneficials, and it is also not possible to detect impacts or even positive attributes of unsprayed Bt plots.

To confirm that scale and reinvasion effects may have compromised the WideStrike field test data package, at least in Arizona, the Panel noted that chlorpyrifos was used in the nontransgenic control. This material is very toxic to natural enemies, including spiders, and the Agency should reasonably have expected significant reductions in the non Bt plots as a check that the experiment had sufficient sensitivity to discriminate treatment effects.

The Panel noted that the numbers of organisms in the Louisiana study seemed to be extremely low, and this requires explanation if data are submitted for review in the form of a final report. The Panel suggested that the numbers were too low to discriminate treatment effects.

The Panel also noted that the current codification of the risk assessment approach does not allow some important positive issues to be highlighted. An important advantage of the use of WideStrike cotton is much lower application rates of chemical pesticides. The early field trial Page 35 of 85 showed, as might have been predicted, that the number of species was higher in the WideStrike field than in the conventional ( non transgenic) cotton culture involving heavier application of chemical pesticides.

With respect to soil degradation, the Panel noted that, strictly speaking, the protein half life study quantifies the temporal change in activity, not of intact protein. If there was direct evidence, then the protein concentration was decreasing. Otherwise, a short sentence qualifying the results would be helpful. Although unlikely, some protein might still be intact but bound or sequestered in some manner such that its activity is not expressed.

Agency Charge 3. The Agency has sufficient information to conclude that there is no hazard from the proposed uses of WideStrike cotton to non target wildlife, aquatic and soil organisms. However, the Agency is requesting additional, primarily long term effects data that were recommended by previous Panels for PIP corn. The supplementary studies would provide additional weight to support the Agency's conclusions.

The Panel is asked to comment on ( a) the scientific value of the proposed additional studies that are identified at the end of the Environmental Assessment section, including avian chronic exposure testing and multi year field and soil persistence/ terrestrial expression studies, and ( b) the applicability of these data to PIP cotton.

Panel Response The Panel was asked to comment on the scientific value of additional studies to assess longer term effects associated with the commercial cultivation of WideStrike cotton. Three aspects were considered: ( 1) additional avian chronic exposure testing; ( 2) multi year field studies to assess potential longer term effects of WideStrike cultivation on persistence of toxins in the soil; and ( 3) populations of non target organisms. The Panel agreed that such studies are of considerable scientific value and finds that with the exception of avian chronic exposure testing, that such studies are applicable to Bt cotton and should be considered. While the Panel is aware of limiting its comments to risk assessment, and not risk management issues, the Panel did not find that such long term studies should necessarily be required as a condition for registration of WideStrike cotton. Overall, the likely reduction in the use of broader spectrum insecticides afforded by the cultivation of Bt cotton in general is likely to have positive effects on the ecosystems. Nonetheless, longer term and broader scale evaluation of PIP crops will be necessary for improved ecological understanding. The Panel believed strongly that long term field studies should not proceed without some guidance relative to experimental protocols and clearly defined endpoints. Without such guidance, we are unlikely to resolve any unexpected detriments or benefits associated with the use of transgenic crops.

Avian Chronic Exposure Testing Page 36 of 85 The Panel found that testing of multi year field effects on non target vertebrates is generally essential. It can readily be seen from the numerous publications Brewer et al. ( 1989, 1989a, 1990, 1992; Tank et al. 1992, 1992a) that significant annual differences are apparent with the same pesticide treatments by the same applicators in the same fields. However, Tier 1 testing of representative vertebrate models shows that the Bt toxins found in WideStrike cotton have no measurable short term adverse effects. Furthermore, direct exposure to Bt toxins through direct ingestion of seed cotton or through ingestion of intoxicated arthropod prey by avian species in the field is likely to be minimal because birds rarely forage in cotton fields ( September 21 24, 1999 FIFRA SAP Meeting). Thus, the probability of direct chronic exposure to Bt toxins in cotton fields is unlikely. Indirect effects on avian populations are possible through the elimination of caterpillar prey. However, since foraging in cotton is minimal, this effect is unlikely to be important. As a result, the Panel finds little scientific merit in conducting additional chronic exposure testing.

Soil Persistence Bt toxins find their way into the soil either through root exudates or the breakdown of crop residues ( shoots and roots). The Panel agreed that data submitted relative to soil persistence suggest that Cry1 proteins are quickly degraded. However, persistence of the Cry proteins goes hand in hand with persistence of crop residues in which they reside. The registrant conducted tests on toxin persistence in two ways; ( 1) by incorporating purified Cry1Ac and Cry1F alone and in combination into soil and ( 2) by incorporating finely ground WideStrike plant material into a cotton soil. In both cases, the ability to detect the toxin was monitored as was the decline over time of the toxicity to a target insect.

The Panel had several comments about this approach. First, the Panel found that incorporation of purified toxins serves only to demonstrate that the toxins are biodegradable. It does not reflect what is likely to happen in the field, nor are half life values derived from these data meaningful. The Panel agreed that, provided it is accessible, the toxin will be broken down. Second, use of finely ground plant material does not reflect actual field situations where residues are incorporated in large ` chunks' that require shredding to initiate decomposition. This discrepancy may be mitigated somewhat by the fact that defoliant use and mechanical harvesting in cotton production does in fact generate reasonably pulverized material after mechanical harvesting is complete. Finely ground plant material obviates the role and possible effects on shredders and also provides a vast surface area for microbial attack thus leading to enhanced rates of decomposition. Access to the toxin by proteases is key to its decomposition. If the toxin is bound in residues, sequestered in soil aggregates or bound to clays or humic material, it is not accessible and hence will accumulate or at least enjoy longer term persistence in the soil. This is likely to be ecologically unimportant as a toxin or toxicant bound in such a manner to resist biodegradation is unlikely to be bioavailable to most soil organisms ( Anhault et al. 2000; Awata et al., 2000; Gevano et al. 2000, 2001; Morrison et al. 2000).

A number of studies have shown that Cry toxins from Bt resist microbial degradation by binding to clay and humic acid fractions in soil, but retain insecticidal activity despite being bound Page 37 of 85 ( Venkateswerlu and Stotzky, 1992; Tapp and Stotzky, 1995; Koskella and Stotzky, 1997; Crecchio and Stotzky, 1998; Tapp and Stotzky, 1998; and Saxena et al., 1999). Further, Saxena and Stotzky ( 2000) showed that larvicidal activity increased with the length of time Bt corn plants were grown, suggesting that microbial degradation may not have kept pace with the increased root exudation of the toxin into soil with time. An alternative explanation may be that the toxin was unavailable for degradation as it was bound, and concentrated with time on the surface of clays and humic materials.

The Panel found that the fate of the toxin in different soil types with different charge characteristics is needed. Studies to understand the mechanism of toxin binding in relation to access of proteases is also important. There is little information on the fate and persistence of Bt toxins in the field, with the exception of one field study reporting that while the levels of Cry1Ab toxin in no till corn decreased to 0.3% of the initial concentration after 200 days, degradation was initially delayed and lower in a conventionally tilled system ( Zwahlen et al., 2003a, b). However, Head et al. ( 2002) did not detect Cry1Ac toxin in soil samples taken about 90 days after the last planting from fields planted with Bt cotton for several consecutive years. The Panel agreed that more data are clearly needed here as this is also a point of ecological as well as public concern. These findings are consistent with and largely echo the concerns expressed by the August 27, 2002 SAP and the current Panel fully agree with the recommendations forwarded by this prior SAP for future multi year testing. The current Panel further suggested that additional studies should consider the role of residue degradation, residue dry matter partitioning and major pools of structural carbon, and measurement of effects on residue shredding organisms.

Non Target Effects Not withstanding some of the flaws outlined under Question 2 to this Panel, Tier 1 testing under current guidelines has demonstrated no measurable short term hazard from the two Cry toxins expressed in WideStrike cotton to representative organisms from terrestrial, soil and aquatic systems. In addition, Bt Cry toxin proteins are susceptible to metabolic, microbial, and abiotic degradation once they are ingested or excreted into the environment. As a result, unlike many insecticides that persist and accumulate through the food chain, Cry proteins are not expected to have the potential for bioaccumulation.

Thus, the Panel agreed with the Agency's conclusion that the Cry toxins in WideStrike cotton pose no short term hazards. However, the Panel also agreed with the Agency that Tier 1 testing is insufficient for assessing the potential for long term environmental effects. Assessing this level of hazard will require carefully designed field studies conducted over longer periods of time and at relevant spatial scales. The bulk of evidence ( Groot and Dicke 2002, Conner et al. 2003) suggested that most of the effects, if any, from Bt toxins in the field will be either sublethal and/ or result largely from indirect effects via complex community interactions. Tier 1 is limited to direct exposures and largely measures only lethal effects. Sublethal effects are usually subtle but may have significant effects on longer term population dynamics and community level interactions ( Elzen 1989; Croft 1990). Examples include reductions in adult longevity, reductions in reproductive output, increased susceptibility to other environmental factors, and altered searching Page 38 of 85 behavior in the case of predators or parasitoids. Although some of these factors could be examined in laboratory studies, their implications and impacts can only be realistically evaluated with longer term field studies. Indirect effects are more complex and likely to be more pervasive due to the large number of interactions. As an example, the soil food web represents an intricate set of feeding relationships.

To evaluate the risk to these organisms of adverse effects of the stacked Cry1Ac  Cry1F genes requires a consideration of the hazard these toxins present and the level of exposure the organisms are likely to have. Extant research has shown that there is no direct toxicity to soil bacteria and fungi, rather, these are the organisms responsible for active degradation of the Cry toxins in soil. Higher order feeders such as protozoa, nematodes, collembolans, mites and earthworms have no receptors for toxin binding and hence are unlikely to be adversely affected by the presence of the toxin in the plant tissues being broken down and thus no hazard. Field evidence with Bt corn supports this point ( Devare et al. 2004).

In general, the evidence presented by the registrant and evaluated by the Agency for collembolan and earthworms also indicated a lack of direct toxicity. However, the Panel questioned the veracity of the collembola data, because the first two tests on the Cry1Ac pure toxin indicate an effect on collembola fecundity, which was reduced by close to 50%. When the two purified toxins, Cry1Ac and Cry1F, were tested together or plant tissue expressing the Cry1Ac toxin were fed to collembolans, no adverse effects were detected. The report submitted to the Agency for review suggested the Cry1Ac toxin may have been contaminated in some way. If the Cry1Ac preparation used was compromised, why was the reduced fecundity not also observed in the combined test? Long term adverse effects are not precluded by these results. Changes in the biological quality of soil accrue slowly. Even in conservation agriculture, no till systems and legume rotations, benefits may not be detectable for 5 10 years. In order to have confidence that WideStrike does not have adverse ecological effects, long term, post release monitoring is needed. As already noted, effects of the toxin may be sublethal or cumulative and the possibility of this needs to be ascertained.

One aspect of crop assessment that has not been dealt with adequately in the current analysis was whether there are changes in dry matter partitioning or carbon allocation within the WideStrike cotton plant tissues that could influence the decomposition dynamics of the crop residues after harvest. The soil microbial community relies on fixed C in plant material for energy and nutrients for metabolism, growth and reproduction. Different forms of C are more or less readily decomposed. Simple sugars and amino acids represent very labile C pools, whereas cellulose and hemi cellulose are more stable pools and lignin the more recalcitrant pool. Should the incorporation of the Cry genes lead to unintended changes in the way that structural C is allocated within the plant, then decomposition dynamics of the material could be altered. An increase in lignin, for example, would result in a slower rate of decomposition. This was a result reported by Stotzky ( 2000).

Stotzky ( 2000) reported that CO2 evolution from soil amended with ground Bt corn biomass expressing the Cry1Ab gene was significantly lower than that from soil containing unmodified, Page 39 of 85 isogenic corn biomass. While the mechanism by which Cry1Ab might depress microbial activity is unclear, unintended changes in structural C allocation within the plant is a possibility and Saxena and Stotzky ( 2001) went on to conclude that increased lignin content in the corn was responsible for their observations. Stotzky has also reported that finely ground Bt rice, potato, and tobacco residues degrade more slowly than their non Bt counterparts in laboratory studies for reasons that are unclear ( Stotzky 2002), as no differences in lignin content were detected for these other crops. Analyzing transgenic crop residues for proportions of structural C in different forms may be useful for addressing concerns for potential differences in the rates of residue decomposition between Bt crops and their non Bt isolines. The relevant comparisons would be: ( 1) insect affected nontransgenic cotton, as C partitioning is grossly affected by insect feeding; and ( 2) insecticide protected non transgenic cotton, the commercial standard. Once structural C forms have been quantified, a soil organic matter dynamics model such as Roth C or the Century model could be used to predict if any change in decomposition dynamics would be expected. The Panel noted that finely ground plant material is not an appropriate means to assess decomposition dynamics that might be operating in the field, nor is it the best means by which to evaluate toxin persistence ( as discussed above). This is another reason why long term post release monitoring is important and why it should include an analysis of the fate of the cotton residues under commercial field conditions.

In and of itself, slower decomposition of residues is not a bad thing. Conservation agriculture strives to increase soil organic matter content and slower decomposition might serve to be beneficial for overall soil quality. However, a build up of crop residues may also be indicative of non target toxicity to a key group of soil decomposers called the shredders. These are microarthropods for the most part, mites being a prime example. Adverse effects on mites could be quite destabilizing to the soil food web, and an effect such as this might not be picked up without longerterm field monitoring. Some consideration should be given to using mites as a target group for toxicity testing due to their important role in communinution.

Overall, long term testing on relevant scales will only be possible once registration is granted and WideStrike can be grown commercially. The Panel recommendations for long term evaluation are consistent with the spirit of recommendations made by NRC ( 2002) and several previous FIFRA SAPs ( e. g., December 1999, August 2002). The NRC, for example, envisioned long term field testing of non target effects and other environmental and pest management concerns as part of what they call " postcommercialization validation testing". The main thrust of such studies would be to conduct appropriate, hypothesis driven, and adequately replicated field studies in large commercial scale plots for the purpose of determining the accuracy and adequacy of pre commercialization testing. This same general reasoning was expressed by the August 2002 SAP. A critical, and still unresolved issue that has been put forward by both the NRC ( 2002) and prior SAPs is the lack of guidelines for conducting long term field studies such that these studies will provide meaningful and robust results. For example, ( 1) which methods and types of data should be collected; ( 2) what spatial scale should be examined; ( 3) which experimental designs are most appropriate; and ( 4) most importantly, what criteria will be used to determine impact or the lack thereof? The Panel believes that long term field studies should not proceed without some guidance relative to protocols and clearly defined endpoints. Page 40 of 85 The Panel recognized that field studies have been and will continue to be conducted to examine non target effects. A number of field level studies have been published on transgenic corn and cotton expressing various Cry proteins in the last few years ( e. g., Candolfi et al. 2004; Musser and Shelton 2003; Men et al. 2003; Al Deeb et al. 2003; Al Deeb and Wilde 2003; Dively and Rose 2002; Moar et al. 2002; Naranjo 2002 and Devare et al. 2004). In general, these studies largely support Tier I hazard testing results reported to the Panel and demonstrate no measurable adverse effects. Several of these studies have been relatively long term (> 3 years). However, in light of the issues raised by Jepson ( 2002) and previous FIFRA SAPs regarding scale, design and sampling issues, it remains unclear whether these studies would be considered sufficiently robust to definitively address population and community level effects if such effects exist. Nonetheless, scientists are using the growing body of data to address some of the issue of concern to the Agency and many of these are being conducted independent of registrant needs. It seems likely that these types of studies will continue to be conducted with or without the support of industry and it would be prudent if the scientific community could provide guidelines that would ensure and perhaps improve the quality and value of the results for both risk assessment and broader ecological understanding. Issues include plot sizes, replication, sampling methods, sampling intensity, appropriate positive and negative controls, taxonomic coverage and clearly specified endpoints. The Panel suggested that some of this guidance may be extractable from existing and ongoing studies.

One of the more significant aspects of field testing that needs to be addressed is spatial scale. Jepson and colleagues ( Jepson and Thacker 1990; Sherratt and Jepson 1993; and Jepson 2002) provide compelling evidence that spatial and temporal components must be considered in evaluating toxicological effects of insecticides and that this is largely driven by the mobility of the species under consideration and their phenology and ecological requirements. While the Panel believed that it would be desirable to conduct studies at spatial scales mimicking commercial production practices, it may not always be feasible to do so based on economic and logistical considerations. Directly working in commercial fields often removes an important level of control necessary to implement a consistent experimental design. Insecticides needed for other pests and other production constraints may interfere. On the other hand, establishing independent commercial scale research plots may be cost prohibitive, especially if studies are to be conducted with sufficient replication. Very large plots may provide the necessary independence, but also introduce significant within plot variation thereby requiring additional sampling for precise estimation ( Jepson 1994). Although more commercial scale study plots may be desirable, the issue of independence versus heterogeneity will need to be assessed relative to plot size. How large do plots really need to be? This will likely be system specific and depend on the taxa being assessed. For example, relatively small plots (< 0.5 acres) may be sufficient for studying some predatory arthropods. Naranjo ( 2002) found that 0.25 acre plots were sufficient to determine differences in a positive control of broad spectrum insecticides for a complex of 20 arthropod predator taxa, indicating that there is a fairly high degree of fidelity within plots of this size. Prasifka et al. ( 2004) has shown that Hippodamia convergens readily move from sorghum to cotton on a commercial scale, but they also showed fidelity of this beetle in cotton even in the absence of its preferred aphid prey. Many of the predatory arthropods found in cotton are generalist feeders and are relatively insensitive to the densities of any one prey. In the western US, pink bollworm, the main target of Bt cotton, is essentially invulnerable to natural enemies. Page 41 of 85 Thus, the removal of this insect from the system has little effect on natural enemy populations. Other Lepidoptera in this area are relatively rare and their presence is inconsistent. Abundant prey in the form of thrips, whiteflies and Lygus bugs are unaffected by Bt cotton. Thus, predators may not be affected by changes in prey density that may cause them to disperse.

Replication is another critical component of the experimental design as it bears directly on the issue of statistical power, or the ability to detect differences that may be relatively small between Bt and non Bt plots. This aspect will clearly be system specific. Perry et al. ( 2003) estimated that a sample size of 60 may be necessary to measure environmental effects of herbicide tolerant crops in the UK, and higher sample sizes were suggested by NCR and the August 2002 SAP. In a recent meta analysis of a 5 year study, a sample size of 40 was only sufficient to discern a 20% significant difference between densities of arthropod predators in non Bt versus Bt cotton expressing Cry1A. In the long run, there will likely be a trade off relative to plot size and replication and this will need to be determined on a case by case basis depending on the taxa to be examined and the ecological setting. In addition, it may be possible to perform retrospective power analyses on many of the past and ongoing non target studies that would be useful in developing guidelines for future studies.

Sampling methods are another important consideration. For example, sweep nets and beat clothes are a common method of measuring relative abundance of foliar dwelling arthropods in row crops such as cotton, and these methods are probably sufficient for comparative studies. Absolute sampling methods such as whole plant counts would be desirable, but such methods are likely to be much more costly due to aggregated arthropod distributions. In the supplemental multi year field studies being requested by the Agency, protocol suggests the use of sweep nets, sticky traps and pitfall traps to assess effects on non target arthropods. These methods will likely produce useful data, but careful consideration will need to be given to the number of samples collected in each experimental unit. The interplay of plot size, replication, sampling method, and sample size for these methods will ultimately determine the statistical power and the ability of the assessment to discern relevant differences.

Taxonomic resolution is another aspect that is critical to address. Many of the existing non target field studies have focused on selected taxa. Such an approach is generally necessary as there are well over 500 species of arthropods alone inhabiting cotton across the southern tier of the US, and the soil invertebrate community is large and diverse as well. Predaceous and parasitic natural enemies have been the focus in many studies of Bt crops. Such species are consistently present in the cotton system; many occur at moderate to high densities, and further, their standing in higher trophic levels may provide insight into effects in lower trophic levels and may act as sensitive indicators of community change. The Panel suggested that assessment of such taxa may be useful for multi year testing. The supplemental studies by the registrant provided relatively broad taxonomic coverage. In particular, the study in Maricopa, Arizona attempted to examine all above ground taxa. Such studies complicate analyses, and careful thought will need to be applied in interpreting the meaning of any observed differences. The resolution of a large percentage of the taxa will likely be too poor to enable definitive conclusions. However, the Panel recognized that studies like this may be helpful in narrowing the taxonomic coverage of future, more targeted evaluations. The choice of taxa to examine may be system specific and should include consideration of likely exposure to Bt toxins through either direct or indirect Page 42 of 85 means.

Final considerations include the inter related issues of study length and desired end points for assessment. With clear focus on short term hazard, Tier 1 testing generally uses direct mortality within a short period of time as the designated endpoint. Such simple endpoints are not likely to be useful, or even measurable, in long term field assessments for environmental effects. Instead, effects ( either positive or negative) are likely to be manifested as reductions or increases in population densities of specific taxa, changes in diversity, or changes in community organization and function. As indicated above for the soil system, such changes may be slow and take many years to shape observable changes. However, even when changes do occur, what criteria should be used to judge a positive, negative or neutral effect? For example, a five year study on the non target effects of Cry1A expressing cotton on 20 arthropod predator taxa ( Naranjo 2002, in part) showed a general pattern of population reductions in Bt cotton. Changes in this select community in Bt cotton varied from a negative change of 19% in one year to a positive change of 15% in another year when compared to non Bt cotton. Irrespective of statistical significance, the predator community declined by 8% averaged over the entire 5 years. This average value would have been different after 2, 3 or 4 years and would likely be different if the study proceeds another 5 years. In contrast, the positive control treatments based on use of broad spectrum insecticides caused an average reduction of 62%. During this same period, functional studies on the natural enemy community showed no statistically significant differences in rates of predation and parasitism between unsprayed Bt and non Bt cotton on two different pests. Irrespective of statistical significance, rates of predation on pink bollworm eggs and pupae declined 4 6% on average in Bt cotton; parasitism and predation of whitefly nymphs increased 5 8% on average in Bt cotton. Has this system been impacted either positively or negatively by the use of Bt cotton?

Horizontal Gene Transfer Some concern has been expressed over the potential for transgenes to move from Bt crops to soil microorganisms. In this regard, we should consider the likelihood of this occurring in the ` bestcase scenario   that is between closely related bacteria in the soil and contrast this with the probability presented by genes located in the plant genome. There is sufficient genetic similarity within this clade to question whether B. cereus and B. thuringiensis are distinct species because what defines B. thuringiensis as distinct from B. cereus is the presence of the plasmid in Bt that contains the Cry toxin and associated cytotoxic genes. If the plasmid is transferred to B. cereus, it becomes indistinguishable from Bt. How frequently this plasmid is exchanged among members of this clade in soil is unknown. B. anthrasis is closely related, but distinguished by carrying a different plasmid with a different set of toxin genes. Recombination or lateral gene transfer between Bt and B. anthracis has not been reported. Thus in nature such gene flow appears to be restricted even between very closely related species. Given this, what is the likelihood that a full length toxin gene will be transferred intact to a recipient bacterium? First, the needed flanking insertion regions would need to be present in the recipient, which in itself, is highly unlikely. Second, there would need to be sufficient selective pressure operating to drive the acquisition of the genes by the recipient bacterium. Such selective pressure is highly unlikely in the soil environment is. Whilst it cannot be completely ruled out, the likelihood of Bt genes transfer from Page 43 of 85 a transgenic crop to soil is exceedingly small. Page 44 of 85 WideStrike Cotton Insect Resistance Management Agency Charge 1. Dose. Three methods ( two laboratory and one field) outlined by USEPA's Scientific Advisory Panel ( 1998) were used to demonstrate that WideStrike cotton expresses a high dose of Cry1Ac and Cry1F against tobacco budworm ( Heliothis virescens, TBW). Dow AgroSciences ( Dow) employed one laboratory based and one field based method to demonstrate that WideStrike cotton has a high dose ( Cry1Ac only, Cry1F is non toxic) against pink bollworm ( Pectinophora gossypiella, PBW) Results of two field studies indicate that WideStrike cotton produces a moderate dose against cotton bollworm ( Helicoverpa zea, CBW), but a very high level of control ( 94%). The Agency concluded that WideStrike cotton expresses a high dose of Cry1F and Cry1Ac against TBW ( Cry1Ac alone expresses a high dose and Cry1F a nearly high dose); a moderate dose of Cry1F and Cry1Ac against CBW, and a high dose of Cry1Ac against pink bollworm.

The Agency asks the SAP to comment on the Agency's analysis of dose for TBW, CBW, and PBW, the likelihood that resistance will be inherited as a recessive trait, and its impact on insect resistance management for WideStrike cotton.

Panel Response The Panel addressed this question with an understanding that a combination of field and laboratory studies performed by the registrant had evaluated the toxicities of the Cry1Ac and Cry1F expressed in WideStrike cotton against TBW, CBW, and PBW. The Panel also provided a detailed response of field efficacy and high dose considerations.

The combination of the laboratory and field approaches clearly demonstrated that the combined expression of Bt proteins in WideStrike cotton meet the Agency's definitions of high dose for PBW and TBW. Based on existing knowledge of the reduced susceptibility of CBW to Cry1Ac, the registrant did not pursue the full spectrum of high dose studies on CBW, but still managed to show good field control relative to non Bt cotton. In general, there was good replication within trials, and with some tests, replication across states, altogether building a robust data set in support of the contention by the registrant that toxin expression in WideStrike cotton constitutes a high dose for PBW and TBW.

One caveat with the field efficacy trials was that 9 of 19 trials were conducted using artificial infestation techniques in the trial plots. While the success of this approach was gauged in some trials by substantial infestations that resulted in the unsprayed control plots, and is often required to obtain a sufficient infestation in the field, the general practice of using laboratory colonies in field studies should be avoided when possible due to concerns about reduced vigor and fitness in frequently inbred lab strains. The potential risk is that genetically compromised lab strains may not provide the full challenge to a breeding line as do natural field populations, and therefore may not represent a full test of the resistance in the breeding line. In future Page 45 of 85 considerations of such data, the Agency may want to consider guidelines for use of laboratory colonies to better guarantee quality control. Poorly managed colonies have a greater probability of producing results consistent with a high dose.

Having established the high dose trait of WideStrike cotton against PBW and TBW, and given the broad history to date of resistance being recessive wherever resistant larvae can survive on Bt transgenic hosts ( Tabashnik et al. 2003), the Panel concluded that it is valid to assume that resistance will likely be inherited as a recessive trait. CBW is more tolerant of both proteins and it seems possible that resistance will be less recessive. Without abundant alternate host plants that do not express these same Bt genes, CBW would be more prone to resistance.

Based on these assumptions and on the criteria to determine functional dominance established by previous FIFRA SAPs, the combined expression of Bt proteins in WideStrike cotton meets the Agency's definitions of high dose for PBW and TBW. In addition, reasonable doses of the combined protein were evident for control of CBW. Based on the high dose evidence, the Panel concluded that it is valid to assume that resistance occurring in PBW or TBW will likely be inherited as a recessive trait. However, CBW is more tolerant of both proteins and it seems possible that resistance will be less recessive. These studies support the position for durable sprayable Bts in organic cotton production and other crops, such as tomatoes. The same high dose/ refuge strategy practiced thus far as a resistance management approach for Bollgard cotton should be applied for WideStrike.

WideStrike cotton does appear to offer a high dose for TBW, a high dose of Cry1Ac for PBW, and reasonable doses of Cry1F and Cry1Ac for CBW. The same high dose/ refuge strategy practiced thus far as a resistance management approach for Bollgard cotton should be applied for WideStrike.

Field Efficacy Five field efficacy trials each were completed for TBW and CBW while a single trial was run for PBW in Arizona. In addition to these three principal pests, field efficacy data was also collected for 5 other cotton pests ( Dow Agrosciences submission: " Efficacy of Cry1F/ Cry1Ac Cotton Against a Wide Range of Lepidopterous Pests", 2002). By measuring cotton boll damage and infestations, the collective field data were conclusive in establishing that WideStrike cotton provided superior control of TBW and PBW relative to either sprayed or untreated plots of the conventional cotton line PSC355. In the case of CBW, boll damage/ infestation levels in MXB 13 were equal to or less than the unsprayed control in 96% of the comparisons ( 53% significantly less damaged) while 58% of the comparisons showed equal or less damage than the sprayed control. Although some trials were conducted in 2001 of two and one gene plants that showed lower efficacy, these trials were believed to be from early selections that likely had some nonexpressing plants.