BCS submitted acceptable data waiver rationale (MRID 47871603) to justify that testing with wild mammals is not necessary. As described above in the risk assessment of Cry1Ab in T304-40 and Cry2Ae in GHB119 cotton, acute oral toxicity studies with mice are available, and the Agency has no evidence to suggest that laboratory mammals are insufficient surrogates for determining toxicity to wild mammals. See the discussion of synergism and protein expression equivalence below; if protein expression is shown to be equivalent between TwinLink(TM) cotton and the T304-40 and GHB119 parent lines, then bridging these data to TwinLink(TM) is acceptable.

		c. Freshwater animals

Data waiver rationale (MRID 47871603) was submitted to fulfill the data requirements for freshwater fish for Cry1Ab and Cry2Ae in TwinLink(TM). Justification rested primarily on the lack of expected exposure of aquatic environments to Cry proteins produced in cotton, and this rationale was determined to be sufficient. BPPD previously determined that exposure to Cry proteins in aquatic environments as a result of registered uses of transgenic cotton is very low (e.g., see US EPA 2008a), and the data waiver rationale was consistent with BPPD's conclusion.

Toxicity data with Daphnia are available for both Cry1Ab and Cry2Ae for use in the TwinLink(TM) risk assessment. Results of these studies are presented above.

		d. Estuarine and marine animals

The Agency typically waives toxicity studies with estuarine/marine fish and invertebrates for Bt Cry proteins; however, the applicant submitted data waiver rationale (MRID 47871603) to fulfill data requirements for estuarine and marine animals. The waiver rationale presented was the same as that discussed above for freshwater fish and was determined to be sufficient based on the Agency's conclusions about exposure in aquatic environments.

   	e. Terrestrial and aquatic plant species

The Agency typically waives nontarget plant testing for Bt Cry proteins, since the active ingredient is an insect toxin (Bt -endotoxin) that has never shown any toxicity to plants. The applicant submitted acceptable data waiver rationale (MRID 47871603) that was consistent with the Agency's position on nontarget plant risks to Bt -endotoxin.

   	f. Invertebrate species

A 28-day study with collembola (Folsomia candida) was conducted to determine the effects of TwinLink(TM) plant tissue on this soil invertebrate (MRID 47640010). Collembola (Folsomia candida) were provided diet containing nominal concentrations of 5.0, 20, or 50% transgenic lyophilized TwinLink(TM) cotton leaf. The test also included a negative control group of collembola fed diet without lyophilized TwinLink(TM) cotton leaf, a control substance group fed diet containing 50% lyophilized nontransgenic Coker 315 (near isoline) cotton leaf, and a positive control group fed diet containing thiodicarb. Mortality in the 5%, 20%, and 50% TwinLink(TM) test groups was 12.5%, 10.0%, and 7.5%, respectively, while cumulative number of offspring per female were 42.2, 41.7, and 33.4, respectively. Mortality in the negative control and Coker 315 groups was 2.5% and 5.0%, respectively, and cumulative number of offspring per female was 26.9 and 38.6, respectively. Mortality and number of offspring were not statistically significant different among any groups tested. Response in the positive control group was as expected. The results of the study show that exposure to transgenic TwinLink(TM) cotton leaves at 5.0, 20, and 50% concentration in the diet has no significant effect on survival or reproduction of Collembola.

A study was also conducted to determine the potential dietary effects of TwinLink(TM) pollen when administered to 2-3 day old honey bee larvae (Apis mellifera, MRID 47640009). Honey bee larval cells were treated with 10 uL of a 30% solution containing 2 mg of TwinLink(TM) pollen. Additional treatment groups included: 1) a negative control (pollen from Coker 315) in combination with 30% sucrose; and 2) a positive control (potassium arsenate in 30% sucrose at 2,000 ug/mL). The amount of pollen administered to the larvae was approximately the maximum amount that is typically fed to honey bee larvae (Babendreier et al. 2004). Emergence was complete on Day 15 of the study. Larval survival and adult emergence was determined (96.25% for TwinLink(TM); 97.5% for the Coker 315; and 0% for the reference substance). Based on statistical analysis, there were no significant differences in survival between the TwinLink(TM) treated larvae and those treated with Coker 315 pollen.

Additionally, if bridging criteria are satisfied, then data developed for the Cry1Ab and Cry2Ae proteins expressed individually in Event T304-40 and Event GHB119 cotton plants, respectively, can also be used for the ecological risk assessment for TwinLink(TM).

	g. Supplemental Information

BCS submitted a summary of nontarget organism testing with Cry1Ab and Cry2Ae (MRIDs 47640008), which summarized the toxicity information and data waiver rationale for these proteins and TwinLink(TM) and included an assessment of ecological. This summary was determined to be supplemental, but provided little additional information for use in the risk assessment.

	2. Synergism of Cry1Ab and Cry2Ae proteins

A sensitive-insect dose-response bioassay with Heliothis virescens neonate larvae was performed to determine whether Cry1Ab and Cry2Ae expressed in TwinLink(TM) cotton plants exhibit synergistic toxicity. Leaf material from the single event cotton plants expressing either Cry1Ab or Cry2Ae and the combined event (TwinLink(TM)) cotton plants were fed to the larvae at multiple concentrations determine the LD50s for the proteins within each plant. Expression of each protein was determined to be equivalent between the TwinLink(TM) plants and the individual parent lines. Based on the predicted and actual LD50s for the proteins in each type of plant, a conclusion of no synergism was made. Therefore, the results of toxicity tests with each individual protein are assumed to adequately represent the toxicity of these proteins when expressed in TwinLink(TM) cotton, assuming equivalent protein expression.

      3. Protein Expression Comparability

Data have been submitted to the Agency showing the proteins expression of Cry1Ab in event T304-40 cotton, Cry2Ae in event GHB119 cotton, and both proteins in TwinLink(TM) cotton. Additional data have been submitted to determine whether the relative potency of plant-produced Cry1Ab and Cry2Ae is similar to the purified bacterially-produced proteins used in the toxicity testing with each individual protein. These data are currently in review. If these data show that each event produced comparable protein expression levels of Cry1Ab and Cry2Ae in each respective parent line as well as the T304-40 x GHB119 hybrid, and that the relative potency of plant-produced and bacteria-produced Cry1Ab and Cry2Ae are equivalent, then bridging from tests for the individual events is appropriate for support of the T304-40 x GHB119 (TwinLink(TM)) registration.

      C. CONCLUSION

As long as BCS shows that Cry1Ab and Cry2Ae protein expression is equivalent between TwinLink(TM) cotton and the individual T304-40 and GHB119 parent lines, then no unreasonable adverse effects to nontarget birds, mammals, aquatic animals, terrestrial and aquatic plants, and nontarget invertebrates will occur as a result of exposure to these proteins from registered use of TwinLink(TM) cotton. This conclusion is based on the environmental risk assessments for Cry1Ab and Cry2Ae proteins expressed individually in Event T304-40 and GHB119 cotton, as well as additional data developed with TwinLink(TM) cotton plant material.

Both Cry1Ab and Cry2Ae have not been shown to have toxic effects on birds, mammals, freshwater invertebrates, terrestrial nontarget invertebrates, and plants, and because exposure is not anticipated in aquatic environments. If protein expression is shown to be equivalent, then the Agency makes a "No Effect" determination for direct and indirect effects to listed species of these taxa for these proteins as expressed in TwinLink(TM) cotton.

E.	References

Babendreier, D., N. Kalberer, J. Romeis, and P. Fluri. 2004. Pollen consumption in honey bee
      larvae: a step forward in the risk assessment of transgenic plants. Apidologie 35: 293-300.

Marvier, M., McCreedy, C., Regetz, J. & Kareiva, P. 2007. A meta-analysis of effects of Bt
      cotton and maize on nontarget invertebrates. Science 316: 1475 - 1477.

National Academy of Science (NAS). 2000. Environmental Effects of Transgenic Plants: The Scope and Adequacy of Regulation is available from the National Academy Press, 2101 Constitution Avenue, N.W., Lockbox 285, Washington, DC 20055;<PHONE_NUMBER> or<PHONE_NUMBER> (in the Washington metropolitan area); http://www.nap.edu.

Romeis, J., M. Meissle, and F. Bigler. 2006. Transgenic Crops expressing Bacillus
      thuringiensis toxins and biological control. Nature Biotechnology 24:  63-71.

Rosi-Marshall E. J., J. L. Tank, T. V. Royer, M. R. Whiles, M. Evans-White, C. Chambers, N. A. Griffiths, J. Pokelsek, and M. L. Stephen. (2007). Toxins in transgenic crop byproducts may affect headwater stream ecosystems. PNAS:  104(41): 16204 - 16208.

Sanvido,O., Romeis, J., Bigler, F. 2007. Ecological Impacts of Genetically Modified Crops: Ten Years of Field Research and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235 - 278.

Saxena, D. and Stotzky, G. 2001. Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biol. Biochem. 33: 1225 - 1230.

U.S. EPA. 1998. "Guidelines for Ecological Risk Assessment."  EPA 630/R-95-002F. Washington, DC, USA. Federal Register, May 14, 1998. 63(93): 26846-26924.

U.S. EPA. 2000. SAP report No 99-06. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Section I - Characterization and Nontarget Organism Data Requirements for Protein Plant Pesticides. Dated February 4, 2000. Available at the EPA website: http://www.epa.gov/scipoly/sap/1999/index.htm#december

U.S. EPA. 2001a. SAP Report No. 2000-07. Sets of scientific issues being considered by the Environmental Protection Agency regarding: Bt plant-pesticides risk and benefit assessments. Dated March 12, 2001. Web site: http://www.epa.gov/scipoly/sap/2000/october/octoberfinal.pdf

U.S. EPA. 2001b. Biopesticides Registration Action Document: Bacillus thuringiensis (Bt)
      Incorporated Plant Protectants. Washington, DC. http://www.epa.gov/opp00001/biopesticides/pips/bt_brad.htm

U.S. EPA. 2002. SAP Report No. 2002-05. A set of scientific issues being considered by the Environmental Protection Agency regarding: Corn rootworm plant-incorporated protectant nontarget insect and insect resistance management issues. Dated November 6, 2002. http://www.epa.gov/scipoly/sap/2002/august/august2002final.pdf

U.S. EPA. 2004. SAP Report No.2004-05. Product characterization, human health risk,
      ecological risk, and insect resistance management for Bt cotton products. Dated August 19, 2004. http://www.epa.gov/scipoly/sap/meetings/2004/june/final1a.pdf

U.S. EPA. 2005a. Biopesticides Registration Action Document: Bacillus thuringiensis Cry2Ab2
      Protein and its Genetic Material Necessary for its Production in Cotton. Washington, DC. http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006487.pdf

U.S. EPA. 2005b. Biopesticides Registration Action Document: Bt Cry1F (synpro) and Cry1Ac (synpro) Construct 281/3006 Insecticidal Crystal Proteins as Expressed in Cotton. http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006512.pdf
U.S. EPA. 2006. The product characterization and human health data submitted in support of the EUP. Memorandum from R. Edelstein, through J. Kough, to S. Matten dated January 27, 2006. U.S. EPA. Washington DC
U.S. EPA, 2008a. Biopesticides Registration Action Document  -  Bacillus thuringiensis modified Cry1Ab (SYN-IR67B-1) and Vip3Aa19 (SYN-IR102-7) insecticidal proteins and the genetic material necessary for their production in COT102 X COT67B cotton, http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006529.pdf

U.S. EPA, 2008b. Biopesticides Registration Action Document  -  Bacillus thuringiensis Cry2Ab2
      protein and its genetic material necessary for its production in cotton. Amended.
      http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006487.pdf

U.S. EPA, 2010. Biopesticides Registration Action Document  -  Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Protectants, http://www.epa.gov/oppbppd1/biopesticides/pips/cry1f-cry1ab-brad.pdf.

U.S. EPA. 2010. Review of Product Characterization Data in support of Sec. 3 Registration of Plant-Incorporated Protectant (PIP) cotton events: GHB119 cotton expressing Bacillus thuringiensis Cry2Ae protein (EPA File Symbol 264-RNOU), T304-40 cotton expressing Bt CrylAb (264-RNOL); and TwinLink(TM) cotton (EPA File Symbol EPA File Symbol264-RNOA), its associated combination PIP product developed by conventional breeding of cotton events GHB119 and T304-40. Memorandum from A. Waggoner, through J. Kough, to S. Bacchus and D. Greenway dated February 4, 2011. U.S. EPA. Washington DC.

U.S. EPA. 2011. Review of Product Characterization and Human Health Data in support of Sec. 3 Registration of Plant-Incorporated Protectant (PIP) event T304-40 cotton expressing Bacillus thuringiensis Cry1Ab insecticidal protein (EPA File Symbol 264-RNOU; OECD Unique Identifier: BCS-GHØØ4-7);  Memorandum from A. Waggoner, through J. Kough, to S. Bacchus and D. Greenway dated November 30, 2011. U.S. EPA. Washington DC.
APPENDIX D: Insect Resistance Management

Cry1Ab, Cry2Ae and their combination (Cry1Ab and Cry2Ae) in TwinLink(TM)

Discussions of EPA's risk assessments for Insect Resistance Management (IRM) are presented in this Appendix. Where deficiencies were identified in the earlier reviews, BCS provided data to satisfy EPA's requirements. Immediately below is the risk assessment completed by EPA's J. Martinez on March 22, 2010. Following this risk assessment are those completed on September 28, 2010 and June 2011.

I. Insect Resistance Management  Review

Below is the BPPD review of BCS' Insect Resistance Management (IRM) plan and supporting data for the application to register TwinLink(TM) cotton PIP as well as supporting documentation for a Natural Refuge request (U.S. EPA memorandum 04/22/10; EPA File Symbol 264-RNOU, MRIDs 4760014 and 476348-23). Deficiencies identified in this application for full commercial registration TwinLink(TM) cotton were satisfied in subsequent submissions in 2010 and 2011. Those risk assessments follow this initial review.

CONCLUSION AND RECOMMENDATIONS

1. DOSE

   a. BPPD concludes that the diet incorporated and overlay assays together (using verification method 1) suggest that TwinLink(TM) and GHB119 may express a high-dose against CBW but that T304-40 may express less than high-dose against the target pest.

   b. BPPD concludes that diet incorporated and overlay assays (using verification method 1) together suggest that events GHB119 and T304-40 may express a high-dose and TwinLink(TM) may express an effective high-dose against TBW.

   c. BPPD cannot make a conclusion about the dose profile for TwinLink(TM) and its events and PBW (using verification methods 3 and 4) because it is unclear how the mortality results reported by Ellsworth and Li (2008) were obtained. When BPPD divided the number of entry holes reported (Tables 9-12) by the number of live larvae and calculated average mortality, the obtained results did not match mortalities reported by BCS.

BPPD's conclusion regarding dose determination (see also Table D.15)
                                     Pest
                                   Method #1
                                   Method #4
                                   Method #3

                                    T304-40
                                    GHB119
                                      TL
                                    T304-40
                                    GHB119
                                      TL
                                    T304-40
                                    GHB119
                                      TL
CBW
                                 Non-High Dose
                              Possible High-Dose
                         Possible Effective High-Dose
                                   Inconcl.*
                                   Inconcl.*
                                   Inconcl.*
                                      --
                                      --
                                      --
TBW
                              Possible High-dose
                                   High-Dose
                              Effective High-Dose
                                      --
                                      --
                                      --
                                      --
                                      --
                                      --
PBW
                                      --
                                      --
                                      --
                                   Inconcl.*
                                   Inconcl.*
                                   Inconcl.*
                                      ---
                                   Inconcl.*
                                   Inconcl.*
* Insufficient information was submitted to the Agency to make a dose determination
** Cry1Ab could not be tested in this experiment because the lab strain was resistant to Cry1Ab.

2. PROTEIN EXPRESSION

   a. BPPD has reviewed BCS' protein expression results reported in their IRM submission and concludes that the single event T304-40 may not express the same levels of toxin as in the dual gene PIP, TwinLink(TM). Event T304-40 expressed higher levels of toxin in leaves of single event plants than leaves collected from TwinLink(TM) plants. For flowers and bolls the pattern was reversed. It is unclear whether these differences in expression results were statistically significant. Because BCS did not provide further information on the differences observed.

   b. Expression of GHB119 in leaves from early to late stage appeared somewhat variable or unstable. Expression levels were measured in leaves in 2004 and 2005 at three developmental stages (early leaf, mid leaf, and late leaf). At developmental stage 1, expression was higher than at developmental stage 2, and at developmental stage 3, expression increased again but to higher levels than observed at developmental stage 1. For early, mid, and late boll, Expression level for GHB119 were also assessed in three different developmental stages of bolls (early, mid, and late). Levels of GHB119 were higher in TwinLink(TM) than in the single event plant during developmental stage 2 and 3. It appears that the difference was approximately two-fold (see Figure 1).

   c. Table D.1 in this review summarizes the protein expression level (ug/g-dry weight) submitted by BCS for T304-40 and GHB119 in different plant tissues. It appears that the same tissue types were lumped together for plants expressing single and pyramided events. Since there appears to be unequal expression of the proteins in plants expressing either the single or pyramided events over the course of the growing season, this approach is not providing the most information. It would be more useful to have the expression data separated by tissue type for each individual event, GHB119, T304-40, and TwinLink(TM), rather than lumped together for the same protein.

   d. BPPD recommends that BCS provide more specific and explanatory information together with their protein expression results in future data submissions for PIP registrations.

3. CROSS-RESISTANCE

   a. BPPD concludes that based on BCS's data there is little concern for cross-resistance between Cry1Ab and Cry2Ae as expressed in TwinLink(TM).

   b. BPPD notes that should resistance evolve in CBW and TBW to Cry2Ae (as expressed in TwinLink(TM) cotton) that Cry2Ab2 (as in Bollgard II cotton) may be compromised as well because, as BCS stated in their report, there is a great potential for cross-resistance between the two toxins.

   c. BPPD notes that the cross-resistance potential is great between Cry1Ab, Cry1Ac, and Cry1F (although somewhat less for Cry1F) based on the competition assays; the three toxins share high affinity binding sites. Should CBW evolve resistance to Cry1Ab, other PIP products expressing Cry1Ac and Cry1F will likely be compromised as well.

 4. MODELING

      BPPD has reviewed the modeling assessment for CBW and accepted the probabilistic approach presented therein. However, BPPD is concerned about parameter assumptions for initial resistance allele and dose mortality that went into the analysis, the lack of including Cry1Ab corn in the modeling landscape, the lack of establishing a high dose for TBW (requested for natural refuge strategy request) and the oversight of not modeling PIP durability for the TBW target pest. These concerns are all addressed in the Deficiencies 2-6 listed below.

5. REFUGE STRATEGY

   a. An IRM plan waiver request for single trait events (T304-40 and GHB119) is acceptable if seeds from those events are not sold commercially and serve only for producing the combination PIP TwinLink(TM). For any other combinations, BCS may need to apply for an experimental use permit or  product  registration. BPPD recommends the use of acreage/geographic limitations for the single trait events as a means to mitigate potential resistance development. BCS should provide information on proposed acreage for these single trait events.

   b. BPPD verified that BCS cited all the relevant natural refuge studies originally developed by Monsanto Company and has obtained a data citation permission letter from Monsanto.

   c. BPPD notes that a second dose verification method is lacking for TBW. Natural Refuge requests were previously granted on the basis that the toxins were expressed at a high-dose against TBW. A second dose TwinLink(TM)-verification method will need to be submitted to support BCS' Natural Refuge request, or BCS can design a model with their specific dose data for TBW.

   d. When BCS has satisfactorily addressed the deficiencies related to the modeling of resistance evolution in CBW and TBW, BPPD will re-assess the natural refuge request for TwinLink(TM) cotton.

   e. If a Natural Refuge strategy is approved by BPPD for TwinLink(TM), the requirements for a non-Bt cotton refuge in conjunction with the planting of any acreage of TwinLink(TM) cotton should remain in place for the following states and regions: Arizona, California, and New Mexico and in the following Texas counties:  Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, Loving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler.

   f. For those States and regions requiring a non-Bt refuge, the following refuge planting requirements should apply:

         i. 5% External, Unsprayed Refuge
         ii. 20% External Sprayed Refuge
         iii. 5% Embedded Refuge

 6. RESISTANCE MONITORING

   a. BCS' approach for developing baseline data for TBW, CBW, and PBW by 2012 may be too protracted in the case that TwinLink(TM) is commercialized before 2012. Baseline susceptibility data should be available by the first growing season of commercial use of the PIP product. BPPD recommends that BCS accelerate the development of these data.

   b. The applicant proposed to monitor for resistance evolution in all three target pests (CBW, TBW, and PBW) in areas of high TwinLink(TM) adoption. BPPD agrees with this proposal but also recommends that monitoring efforts additionally focus on regions in states with little natural refuge.

   c. BCS should follow the monitoring paradigm established for other Bt cotton registrations; this point is addressed in Deficiency 8.

7. REMEDIAL ACTION PLAN
   a. BCS needs to have an established remedial action plan in place for each target pest of TwinLink(TM). This is addressed in Deficiency 8 below.
   b. BPPD notes that it will not be sufficient if BCS "attempts" to confirm and characterize resistance upon receiving notification of unexpected damage. Rather, unexpected pest damage reports should be taken seriously and followed up immediately to conclusively determine whether the cause of crop damage is due to resistance or other reasons. BPPD is aware that it can take months before a determination can be reached.

8. GROWER EDUCATION

   a. It is recommended that BCS develop a grower education program that targets states and counties where a non-Bt corn variety must be planted as the refuge for TwinLink(TM) cotton.

   b. It is recommended that BCS develop a grower guide that at a minimum describes the technology and includes unexpected damage guidelines.

9. GROWER COMPLIANCE

   a. BPPD notes that BCS should have a well developed grower-education program (including grower guide) for all regions with clear indications for regional requirements.

   b. A natural refuge strategy for TBW and CBW would eliminate the need for growers to plant a refuge where alternate hosts are abundant. However, there are cotton-growing regions where natural refuge is not applicable and where planting a refuge is still required. For those regions it is recommended that BCS develop, implement, and report to EPA on programs to evaluate and promote growers' compliance with IRM requirements.

   c. Grower compliance in these regions should be assessed with on-farm assessments in addition to anonymous phone surveys. BPPD recommends that BCS use the existing grower compliance paradigm for other Bt cotton registrations.

10. REPORTING REQUIREMENTS

      BCS stated that "once the baseline susceptibility data was collected for the three primary target pests and a discriminating dose level was established, annual monitoring would be conducted and reported to the agency". BPPD recommends that BCS submit annual reports for the following:
         i) Baseline and discriminating dose work irrespective of their status;
         ii) Sales data;
         iii) Annual resistance monitoring; and
         iv) CAP results for cotton growing regions where Natural Refuge is not applicable.
11. DEFICIENCIES

Deficiency 1: The dose profile reported for PBW cannot be verified by BPPD until BCS clarifies how mortalities were calculated for the experiments using high-dose verification methods (3) and (4).

Deficiency 2: In the modeling analysis provided by BCS, only CBW was modeled but not TBW. For a new refuge strategy such as Natural Refuge, each main target pest needs to be evaluated separately via a simulation model that incorporates biology, genetics, toxicity, and ecology of the target pest. BCS needs to provide simulation modeling for TBW in order to support their natural refuge request.

Deficiency 3: The natural refuge paradigm was approved for Bollgard II and WideStrike with a high-dose profile for TBW. If BCS wishes to pursue the natural refuge strategy for TwinLink(TM), then a second high-dose verification study will need to be submitted to support that the PIP expresses a high-dose against TBW, or BCS can design a model with their specific dose data for TBW.

Deficiency 4: BPPD is concerned with the dose assumptions for CBW and recommends that an additional analysis be conducted using somewhat lower mortality assumptions based on the study results reported by BCS. The bioassay experiments showed that Cry1Ab caused mortality ranging 88.5-100% (see this review). Estimated mortalities from the artificial infestation experiments (2007-2008) ranged from 99.5-99.7% (see this review). However, BPPD cannot verify the results of the artificial infestation experiments due to lack of information provided (i.e. % egg viability). At the moment, there is no evidence that Cry1Ab expresses high-dose against CBW. BCS concluded, too, that TwinLink(TM) expressed less than high-dose against CBW due to the target pest being less susceptible to Cry1Ab and lower level of expression of Cry1Ab in the parental event for TwinLink(TM). Hence BPPD recommends exploring dose mortalities ranging from 90-99% in the simulation model.

Deficiency 5: BPPD is concerned about the most likely value chosen for initial resistance allele frequency for Cry1Ab (0.0002). Although no field resistance to Cry1Ab has been documented yet for corn earworm (aka CBW), the toxin has been in use for over a decade (in Bt corn) and selection may likely have increased the frequency of potential resistance alleles. However, because corn earworm does not overwinter in the Corn Belt, BPPD agrees that the most likely value will be somewhat lower than could otherwise be expected. BPPD recommends that BCS consider modeling a most likely resistance allele frequency of 0.001 for Cry1Ab in CBW.

Deficiency 6: BCS needs to include Cry1Ab corn and Cry1Ab cotton in the modeling landscape when assessing the risk of resistance evolution in the target pests because Syngenta has registered Bt cotton and Bt corn and Monsanto has registered Bt corn products expressing Cry1Ab. These corn and cotton PIPs are grown in the Cotton Belt and need to be included in a simulation analysis for TwinLink(TM).

Deficiency 7: BCS needs to have a well-developed monitoring plan in place before TwinLink(TM) is approved for commercialization (i.e. sampling regions, sampling scheme, etc). BPPD recommends that BCS follow the monitoring paradigm established for other Bt cotton registrations.

Deficiency 8: BCS needs to have an established remedial action plan in place for each target pest of TwinLink(TM). It is not sufficient to provide an outline or general statement about different kinds of approaches. BPPD recommends that BCS follow the existing remedial action paradigm for Bt cotton PIPs.

     I. BACKGROUND  -  Insect Resistance Management

TwinLink(TM) cotton is a pyramided transgenic cotton trait that expresses Cry1Ab (Event T304-40), Cry2Ae (Event GHB119), and a glufosinate ammonium (herbicide) tolerant active ingredient based on LibertyLink technology. TwinLink(TM) was developed by BCS using conventional breeding techniques.

Cry2Ae is a new PIP for Bt cotton that allows for the control of Lepidopteran pests. Small field trial evaluations with artificially and naturally infested plots using the three major target pests Cotton bollworm (Helicoverpa zea, CBW), Tobacco budworm (Heliothis virescens, TBW), and Pink Bollworm (Pectinophora gossypiella, PBW) demonstrated that there was minimal damage to TwinLink(TM) cotton (expressing Cry2Ae and Cry1Ab) during the growing seasons of 2004 and 2005.

Cry1 and Cry2 are effective lepidopteran toxins and share less than 20% amino acid homology. Crickmore et al. (1998) constructed a phylogenetic tree using amino acid sequence identity as homology criteria for all presently known Bt crystal proteins. This tree indicates that Cry1Ab and Cry2Ae are genetically dissimilar entities and share little sequence homology.

Currently there are five other Cry toxins registered in Bt cotton for the control of Lepidopteran pests; those are Cry1F, Cry1Ab, Cry1Ac, Cry2Ab2, and Vip3Aa19. Pyramids of Cry1F and Cry1Ac, Cry1Ac and Cry2Ab2, and Cry1Ab and Vip3A19 are expressed in WideStrike cotton (product of Dow AgroSciences), Bollgard II cotton (product of Monsanto), and VipCot (product of Syngenta), respectively. All pyramided cotton PIP are effective against the three main target pests CBW, TBW, and PBW.

II.       PEST BIOLOGY AND ECOLOGY

TBW, CBW, and PBW are considered the three major target pests for TwinLink(TM) Cotton. Fall armyworm (Spodoptera frugiperda, FAW) and Beet armyworm (Spodoptera exigua, BAW) are considered secondary target pests. A summary of the biology and ecology for major Bt cotton target pest, such as TBW, CBW, and PBW, can be viewed in the IRM section of the Agency's 2001 Bt crop reassessment document at http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm. Since the publication of the Agency document, additional biological and ecological information has become available. BCS summarized new study results (MRID 476400-14, Vol. 15), which are reported below.

                     1.      Cotton Bollworm (Taken from MRID 47640014)

CBW is highly mobile and can move long distances. The general pattern of CBW migration is a northward movement (U.S. EPA, 2001). At the 2001 Scientific Advisory Panel (SAP) the general consensus was that southward movement to overwinter was not proven but that there was good circumstantial evidence. Further research on south migration was done by Gould et al. (2002): they used 13C/12C stable isotope assessment of alternative host used by CBW and showed indirectly that late season CBW moths captured in Louisiana and Texas could be migrants from the northern Corn Belt. Typically, greater than 50% of the moths captured in August through October have isotope ratios indicative of larval feeding on C4 plants (i.e. tropical and prairie grassland plants), even though local C4 crops were harvested at least one month earlier and the authors presume at the Texas trial site that there are also no common wild C4 plants. Cotton, along with wheat, rice, barley and other ancient plants are C3 plants, easily differentiated from C4 plants.

Directly related to migration is the overwintering behavior of the insect. Schneider (2003) studied the overwintering of CBW in cotton fields in the northeast portion of Mississippi. He found that densities of overwintering pupae varied greatly across years; in non-Bt plots densities ranged from 0-67 pupae/ha for CBW. Lower densities of CBW pupae (0-214 pupae/ha) were found over the seven-year study in Bt cotton fields. Only partial control of CBW was possible with these first generation single gene Bt cotton plants expressing only the Cry1Ac protein.

Local temporal and spatial dispersal in CBW (Han and Caprio, 2002) were studied by using polymorphic allozyme and RAPID-PCR makers. For CBW, Han and Caprio (2002) found that population differentiation at local and regional areas was very low, not unexpected for a highly polyphagous and migratory insect. Contrary to TBW, the genetic variance was not significantly different between the four seasonal generations for CBW.

Similarities amongst pheromone trap catches of CBW in eastern North Carolina suggested a high level of CBW moth movement into and out of localized areas and was much more significant than anticipated (Jackson et al., 2003). Cotton production in localized areas has little impact on the number of CBW moths in the local environment.

Gore et al. (2002) monitored larval CBW movement on cotton and compared movement on Bt cotton (Bollgard) and non-transgenic cotton. On pre-flowering plants at 1, 3, 6, and 24 hours after infestation 47.8%, 39.4%, 20.9%, and 13.0% of the larvae, respectively, remained in the terminals of non-Bt cotton plants. For Bt cotton, this was 28.7%, 11.4%, 6.3%, and 1.3%, respectively. A day after artificial infestation, larvae were found 2.4 main stem nodes below the terminals on flowering non-Bt plants, for Bt cotton this was 5.7 main stem nodes below the terminals.

In the Mississippi Delta, moth populations were monitored from 1986 to 2005 (Adamczyk and Hubbard, 2006) with pheromone traps. During the non-crop months, moderate numbers of moths of CBW were typically captured by 20 March. From 1986-1996, the mean number of CBW moths captured per trap and per night was 15.7 with a maximum of 310. Since 1997, CBW populations have declined, with a mean number per trap and per night of 6.2, and a maximum of 70. The decline may due to wide-scale planting of Bt cotton.

Jackson et al. (2004) estimated the number of CBW that developed on conventional and Bt cotton in 2000-2002 for North Carolina. Across 7 locations they found 60,283 4[th] and 5[th] instar living larvae/ha in conventional cotton, which produced 47,078 adults/ha and damaged 367,835 bolls/ha. The Bt cotton (Bollgard event) did not significantly reduce larval numbers, but damaged bolls and adults were significantly less. The numbers of larvae were also estimated in fields utilizing the conventional chemical insecticide regime in 2001-2002; non-treated fields produced 75,510 larvae/ha, while sprayed fields had significantly lower levels, only 28,973 larvae/ha.

Alternative cultivated or wild hosts, which can be used as non-crop or natural refuges, have been extensively studied since 2001. Jackson et al. (2003) studied the larvae, pupae and adult populations on different hosts in eastern North Carolina and found that corn was the major producer of CBW larvae in the early season and stayed a major producer until the last week of August. These results indicated that field corn may serve as refuge for CBW much longer than originally expected. Beginning in mid-August, soybean and conventional cotton became the major crop sources for CBW production. Pupae and adult production followed the same trends.

Kurtz et al. (2004) used these estimates (Jackson et al., 2003) and unpublished data by Jackson in their computer simulation model. For the first two CBW generations, 90% of the larvae developed on corn, 5% on cotton and 5% on soybean. For the third CBW generation in eastern North Carolina the estimates dropped to only 17.39% production on corn, with increases to 36.34% production on cotton, 0.05% on peanuts and 40.99% on soybean. Storer et al. (2001) confirmed these results, finding that CBW adults emerging from ear-stage cornfields infest cotton, soybean and other crops In North Carolina. The level of adult CBW production was greatly decreased (~80%) in Bt corn, while non-Bt corn produced 20-30 adults per 100 ears.

In Louisiana, Peters et al. (2004) monitored larval CBW populations in several field crops during the 2002 and 2003 growing seasons. Larval populations were highest in sorghum: approximately 32,000 larvae/acre in 2002 and approximately 12,000 larvae/acre in 2003; field corn: approximately 20,000 larvae/acre in 2002 and approximately10,000 larvae/acre in 2003. Larval densities on soybean and on non-transgenic and Bt cotton were much lower (<3000 larvae/acre). Larval populations in corn, sorghum, maturity group 6 soybean, cotton and Bt cotton peaked on 25 June, 12-14 July, 2-4 August, 2-4 August, and 11-16 August, respectively. The only crop that exhibited similar temporal synchrony with bollworm larvae on Bt cotton was soybean maturity group 6, indicating the possibility of including it in an IRM strategy. A more extensive study over two years and over five states demonstrated again that non-cotton crops, such as corn, grain sorghum, peanut and soybean, support much larger larval CBW populations than cotton throughout the season. In June and portions of July the larval CBW populations were almost entirely restricted to corn and in July and August were observed in non-cotton crops more frequently and in larger numbers than in cotton. Again it was shown that CBW moths move extensively from their natal host origins. The number of CBW adults captured in pheromone traps at interfaces of fields of Bt cotton and various crop hosts rarely varied among interfaces, except for highly attractive corn (Jackson et al., 2008). Tillman and Mullinix (2004) showed that CBW females preferred to oviposit in grain sorghum over cotton; the number of eggs per plant and percentage of plants with CBW eggs was higher in sorghum trap crop than in cotton trap crop for two years.

Larval development studies in the lab of CBW on sorghum, corn, soybean and cotton showed a survival of 73, 55, 26, and 13%, respectively and a mean development time to pupation of 15.6, 12.4, 18.4, and 25 days, respectively (Gore et al., 2003). Cotton was the least suitable host, which was also reflected in the significantly lower pupal weight. Blanco et al. (2007) showed that over a three year period in Mississippi and Mexico, garbanzo bean, an important legume crop in Mexico, had an annual CBW level of 96,000 larvae/ha on garbanzo bean, and approximately 5 times less larvae on cotton and approximately 6 times less larvae on velvetleaf, respectively. Low densities of adult CBW moths (0-275 ha/yr) emerged from plots and were not significantly different between the three crops. Cover crops, such as legume mixture, crimson and rye seem to be more preferred by heliothines than the cotton crop. In sweeps no heliothine could be caught on the cotton plants; in contrast, around 0.5 heliothine larva/sweep were found on the cover crops (Tillman et al., 2004).

Natural mortality of CBW was studied from 1991-95 in short season cotton in Texas with one generation per year (Sansone and Smith, 2001). Mortality of 71-95% could be consistently expected in the egg and first-instar stages. The more detailed studies in 1991-1993 partitioned the mortality; 3.4% was caused by Trichogramma egg parasitation, larval parasitation rarely exceeded 5%, but numerically the most import mortality factor was egg consumption of around 77% by Orius insidiosus (minute pirate bug).

                     2.      Tobacco Budworm (Taken from MRID 47640014)

TBW is a highly mobile insect that becomes less mobile in the mid to late summer when it prefers cotton as a host plant. Directly related to movement is the overwintering of the insect. Recently, Schneider (2003) studied the overwintering of TBW in cotton fields in the northeast portion of Mississippi. He found that densities of overwintering pupae varied greatly across years: in non-Bt plots densities ranged from 0-303 pupae/ha TBW. No TBW pupae were found over the seven-year study in Bt cotton fields. Using pheromone traps to estimate the total area-wide TBW population overwintering, Schneider (2003) found that in the pre-transgenic cotton era (<1996) cotton fields typically accounted for up to 10% of the overwintering TBW population. After introduction of Bt cotton, but prior to expanded no-till cotton planting in the 1996-2001 era, the estimate was less than 2%.

Local temporal and spatial dispersal of TBW (Han and Caprio, 2004) was studied by using polymorphic allozyme and RAPID-PCR makers. TBW populations overall showed little special differentiation, indicating a highly effective gene flow rate over the spatial scale under investigation (Han and Caprio, 2004). Allelic differentiation arose more frequently in the second generation than in the other three generations, suggesting that TBW moth dispersal was more limited during the midsummer. During midsummer, cotton is the main host available for TBW in the Mississippi Delta. Once cotton flowers, moth movement declines along with the availability of a ready nectar source. Because the populations never reached genetic equilibrium, Han and Caprio (2004) could not determine the extent of movement reduction.

In the Texas High Plains TBW adult populations were monitored weekly from March to November between 1982 and 1995 using pheromone traps at Lubbock and Halfway Texas, prior to the introduction of Bt cotton (Parajulee et al., 2004). The bollworm/budworm complex consisted of 98% CBW and 2% TBW. For weekly moth abundance, a positive correlation was shown with weekly temperature, degree-days and weekly average precipitation, and a negative correlation was shown with the weekly wind velocity. Data from this study indicate that the late-season moth catches could be indicative of the dynamics of the early-season catches the following year.

In the Mississippi Delta, moth populations were monitored from 1986 to 2005 (Adamczyk and Hubbard, 2006) with pheromone traps. During the non-crop months, moderate numbers of moths of TBW were typically captured by April 10[th]. From 1986-1996, the mean number of TBW moths captured per trap and per night was 19.5 with a maximum of 500. Since 1997, populations have declined for TBW with a mean number per trap and per night of 2.9, and a maximum of 25. The decline may be due to wide scale planting of Bt cotton.

In the Southeastern US, the first TBW generation develops almost exclusively on weeds; throughout the whole growing season cotton is a less important host, because of the abundance of other host plants (Abney et al., 2004). Abney et al. (2005) also showed, using 13C/12C stable isotope assessment, that TBW fed consistently on C3 plants (examples of C3 plants are cotton, soybean, clover, and peanut). Unfortunately this technique could not be used to determine the natal origin of the TBW.

Blanco et al., (2007) showed that over a three year period in Mississippi and Mexico, garbanzo bean, an important legume crop in Mexico, had an annual TBW larval density of 287,000 larvae/ha, approximately 4 times higher than in cotton, and approximately 5 times higher than in velvetleaf. Also the TBW moth densities emerging from garbanzo bean (20,180 adults/ha) were approximately 13 times higher than from cotton and approximately 35 times higher than from velvetleaf.

Cover crops, such as legume mixtures, crimson, and rye, seem to be more preferable to heliothines than cotton. In sweeps, no heliothine could be caught on the cotton plants; in contrast, around 0.5 heliothine larva/sweep were found on the cover crops (Tillman et al., 2004).

                     3.      Pink Bollworm (Excerpted from MRID 476400-14, modified by BPPD)

Flight-mill experiments by Wu et al. (2006) showed that over a 72h flight the mean accumulated flight distance and flight duration of PBW were 41.25 km and 23.87 h for females and 23.46 km and 14.12 h for males. For unmated moths, the flight activity reached a peak after 3-5 days after eclosion, and then reduced gradually. Optimum temperature for flight was from 24 to 28°C and the optimum relative humidity ranged from 75 to 90%.

While PBW can be good fliers, local adult movement is limited; in most cases dispersal is limited to <1 km (Carrière et al., 2001a). Besides adult movement, oviposition preference is also important. Liu et al. (2002) found that in greenhouse experiments females did not lay fewer eggs on Bt cotton bolls than on non-Bt cotton bolls. They also revealed that the number of eggs laid per boll was negatively associated with boll age and positively associated with boll diameter; more eggs per boll were laid on plants with more bolls. The distribution of eggs was clumped, which is an indication that boll quality rather than avoidance of other eggs was the primary factor in oviposition preference. Liu et al. (2002) also studied mining behavior of neonate larvae. The number of entrance holes per boll did not differ between Bt cotton and non-Bt cotton. As with female moths, neonate preferred younger bolls and larger bolls.

Carrière et al. (2001c) studied the prediction of spring moth emergence and its use for cultural control of the insect. Accumulation of heat units from January 1st accurately predicted the rate of PBW emergence in different regions in Arizona. Emergence was significantly higher in higher altitude regions. Most moths emerge from diapause too early to reproduce on cotton, a phenomenon known as `suicidal emergence'. Manipulation of the planting date could be a good cultural control, but shortening the growing season can reduce yield.

Most larval stages of PBW are relatively protected from natural enemies because they feed on seeds inside the cotton boll; eggs laid externally on vegetative structures, however, are vulnerable to predation and parasitism. Predation rates on sentinel PBW eggs were compared on Bt and non-Bt cotton plants (Sisterson et al., 2004). Many eggs were attacked by predators with pierce and/or sucking mouthparts and by lacewing larvae. Between Bt and non-Bt plots, no significant differences were found for PBW egg predators; approximately 1.5-3 predators per plant were recovered, and PBW egg predation rates were approximately 40-50%.

In Naranjo (2005), sentinel eggs of PBW also experienced the same rate of predation in Bt and non-Bt cotton. Egg predation rate varied from around 53% in 2001 to approximately 35% in 2003. No parasitism was detected in eggs that survived predation. On average, chewing and sucking predators were each responsible for approximately 50% of the total mortality. Naranjo (2005) also found no significant difference for rates of pupal mortality between Bt and non-Bt cotton. Pupal mortalities of around 40% in 2002 and 64% in 2003 were observed. No parasitism was observed in intact pupae.

III.       DOSE

TwinLink(TM) cotton is a combined trait product made through traditional breeding of events T304-40 and GHB119 which express Cry1Ab and Cry2Ae, respectively. BCS submitted 1) protein expression data for different cotton tissues; 2) LC50 data for Cry1Ab and Cry2Ae and their target pests; and 3) dose information for Event T304-40 (Cry1Ab), Event GHB119 (Cry2Ae), and TwinLink(TM)  using methods #1 and #4 as described by the SAP (U.S. EPA, 2001).

   a) Protein Expression

BCS submitted protein expression results for cotton tissues, which were measured using ELISA based assays in 2004 and 2005. Bayer determined that protein expression in Cry1Ab and Cry2Ae cotton as well as TwinLink(TM) cotton plants depended on the type as well as maturity of tissues assayed (see Figure 1). The lowest expression was found in nectar, pollen, and seeds; the highest expression was observed in leaves, roots, squares (flower buds containing male and female reproductive parts), apex, stems, and bolls (fertilized ovum followed by enlargement, filling, and maturation of structure) (see Table 1). Cry1Ab, as expressed in TwinLink(TM), had consistent expression in leaves and squares with higher expression in bolls and flowers. Cry2Ae, as expressed in TwinLink(TM) had relatively high expression levels in leaves across the growing season and in late bolls (data from 2005). Expression was typically higher in Cry2Ae than Cry1Ab (up to 10X). Cotton plants expressing the single events were analyzed in 2004 and showed similar protein expression levels as the plants expressing both events. BCS concluded that there was no suppression or enhancement of protein expression when Cry1Ab and Cry2Ae were expressed in the same plant.

Figure D.1. Expression of Cry1Ab and Cry2Ae as % Bt protein/total extractable protein - TL denotes TwinLink(TM) (extracted from MRID 476400-14)

Table D.1. Expression of Cry1Ab and Cry2Ae in plant tissues
                                 PLANT TISSUE
                   AVE EXPRESSION LEVEL (ug/g - dry weight)

                                    Cry1Ab
                                    Cry2Ae
Roots
                                   2.9-11.2
                                   8.8-19.7
Stems
                                    0.5-7.3
                                    4.4-7.3
Leaves
                                    0.4-5.2
                                   10.7-63.3
Squares
                                    0.8-3.9
                                   3.4-12.4
Apex
                                    1.0-1.6
                                   6.1-10.7
Bolls
                                    0.2-0.6
                                    3.3-4.9
Flowers
                                   8.7-12.5
                                    4.8-6.3
Seed
                                    0.1-0.7
                                   0.02-1.6
Pollen
                                   0.03-0.07
                                   0.06-0.6
Nectar
                                 0.00001-0.005
                                       0
(Extracted from MRID 476400-14)

   b) LC50 Data for Cry1Ab and Cry2Ae in CBW and TBW
BCS provided results from insect bioassays using purified individual Bt proteins in a surface layer diet for the target pests of TwinLink(TM) cotton. Table 2 shows the mean LC50 values and ranges for six target pests. BCS described the insect susceptibility for Cry1Ab as follows: TBW > PBW > Old World BW (OWBW, Helicoverpa amigera) >> CBW. The susceptibility for Cry2Ae was described as: TBW > PBW >> CBW > BAW > FAW > Old World BW.

Table D. 2. Mean LC50 values and ranges for Cry1Ab and Cry2Ae
INSECTS
                                    Cry1Ab
                                  (ng/cm[2])
                                    Cry2Ae
                                  (ng/cm[2])
Heliothis virescens, TBW
                                      7.2
                                  (4.9-10.5)
                                     4.1
                                   (2.9-5.5)
Helicoverpa zea, CBW
                                     1357
                                  (462-2844)
                                    238.9
                                 (107.7-473.1)
Helicoverpa armigera, Old World BW
                                     78.5
                                 (41.1-149.9)
                                     3556
                                  (2824-4709)
Pectinophora gossypiella, PBW
                                     41.2
                                  (6.9-152.0)
                                     11.6
                                  (4.9-22.2)
Spodoptera frugiperda, FAW
                                 >>2000
                                    1154.3
                                (682.0-1680.1)
Spodoptera exigua, BAW
                                 >>1350
                                     486.4
                                 (336.5-685.5)
 (Extracted from MRID 476400-14)

   c) Dose Determination

The 1998 SAP Subpanel discussed ways to define and measure "high dose" in plants. It was agreed that the definition of high dose as "25 times the toxin concentration needed to kill susceptible larvae" was reasonable based on current empirical data. However, the Subpanel recognized that it was conceivable that a heterozygote might develop with higher than 25-fold resistance. The major problem identified by the Subpanel was in determining if this 25-fold level was achieved in a specified cultivar. After much discussion, it was concluded that there were at least five ways to assess this 25-fold level, but that some approaches were more appropriate for specific crop pests. The Subpanel concluded that a cultivar could be considered to provide a high dose if two of the five approaches described here indicated presence of a high dose. The five approaches generally used for Lepidopteran systems are:

(1) Serial dilution bioassay with artificial diet containing lyophilized tissues of Bt plants (tissue from non-Bt plants serving as controls);

(2) Bioassays using plant lines with expression levels approximately 25-fold lower than the commercial cultivar (determined by quantitative ELISA or some more reliable technique);

(3) Survey large numbers of commercial plants on sentinel plots in the field (e.g., sentinel sweet corn method) to make sure that the cultivar is at the LD99.99 or higher to assure that 95% of heterozygotes would probably be killed. With this approach Bt sweet corn hybrids are used to attract high densities of ECB and cotton bollworm (Helicoverpa zea)(Boddie)) (CBW/CEW)  moths, sampling can be limited to sweet corn ears in the Bt plot (ca. 1/4-1/2 acre block), and a frequency of resistant phenotypes can be estimated as the ratio of density of larvae/plant in Bt sweet corn to density of larvae/plant in an adjacent planting of non-Bt sweet corn (Andow and  Hutchison, 1998; Hutchison, unpublished data);

(4) Similar to (3) above, but would use controlled infestation with a laboratory strain of the pest that had an LD50 value similar to field strains; and

(5) Determine if an older instar of the targeted pest could be found with an LD50 that was about 25-fold higher than that of the neonate larvae. If so, that stage could be tested on the crop plants to determine if 95% or more of the older stage larvae were killed.

BCS applied three methods to test for high-dose of TwinLink(TM) and its single events.

   * Method (1) utilized lyophilized plant tissue from events T304-40, GHB119, and TwinLink(TM) mixed with artificial diet to produce 10X and 25X concentrations. The bioassays were conducted as diet incorporated assays and diet overlay assays with TBW and CBW.
   * Method (4) involved controlled infestations of TwinLink(TM) cotton plots with susceptible lab strain of CBW and susceptible lab strain of PBW to assess the level of mortality.
   * Method (3) utilized a homozygous resistant lab strain (Cry1Ac) of PBW with which young cotton bolls were infested.

High-Dose Verification Method (1):

   1. Cotton Bollworm

Lyophilized plant material from greenhouse grown cotton was mixed into the bioassay diets at different concentrations (4% and 10% of Cry-protein). For the negative control, 0, 0.8, 4, and 10% of non-Bt cotton (Coker312) were used. Twenty-four neonate larvae were exposed at each treatment level (see Table D.3); two replicates per treatment were carried out. Mortality was scored at days 4, 7, 12, 17, 19, 21, and 24, and average mortality was calculated from the two replicates.

For overlay assays, the same leaf material was used and the lyophilized powder was mixed in a 0.2% agar solution. The concentrations tested were 4% and 6.9% of Cry-protein. Liquid diet was poured into each well and allowed to dry. Different concentrations were prepared (see Table D.4) and overlaid onto the solidified diet. Mortality was scored at day 3, 7, 11, 15, 18, and 21.

The applicant reported that control mortality was difficult to manage using cotton leaf tissue due to the presence of gossypol. Diet incorporated assays had higher control mortality (17.5-29.5% on day 17) than overlay assays (16.8-19% on day 15); hence overlay assays were best for verifying TwinLink(TM) cotton's high-dose level. BCS concluded that TwinLink(TM) expressed less than high-dose against CBW due to the target pest being less susceptible to Cry1Ab and lower level of expression of Cry1Ab in the parental event for TwinLink.

Table D.3. CBW mortality to Cry1Ab, Cry2Ae, and TwinLink(TM) - diet incorporated assays
Treatment
                           % Treatment Concentration
                                  % Mortality

                                     Day 4
                                     Day 7
                                    Day 12
                                    Day 17
                                    Day 19
                                    Day 21
                                    Day 24
Coker312
                                      10
                                       0
                                     10.5
                                      17
                                     29.5
                            All pupae or pre-pupae

                                       4
                                       4
                                     10.8
                                      15
                                      23
                            All pupae or pre-pupae

                                      0.8
                                       0
                                      8.5
                                     15.5
                                     17.5
                            All pupae or pre-pupae

T304-40, Cry1Ab
                                      10
                                      21
                                     85.5
                                      100

                                       4
                                       6
                                      21
                                      41
                                      55
                                     57.5
                                     81.5
                                      89
GHB119, Cry2Ae
                                      10
                                      29
                                      72
                                      100

                                       4
                                      19
                                      31
                                      52
                                     73.5
                                     73.5
                                      84
                                      95
TwinLink(TM)
                                      10
                                      44
                                      85
                                      100

                                       4
                                     14.8
                                     55.5
                                      70
                                      94
                                      97
                                      100

Modified from MRID 476400-14

Table D.4. CBW mortality to Cry1Ab, Cry2Ae, and TwinLink(TM) - overlay assay method
Treatment
                           % Treatment Concentration
                                  % Mortality

                                     Day 3
                                     Day 7
                                    Day 11
                                    Day 15
                                    Day 18
                                    Day 21
Coker312
                                      6.9
                                       0
                                     10.5
                                     16.8
                                     16.8
                            All pupae or pre-pupae

                                       4
                                       0
                                       8
                                      17
                                      19
                            All pupae or pre-pupae
T304-40, Cry1Ab
                                      6.9
                                       0
                                     35.5
                                      54
                                     88.5
                                     88.5
                                     88.5

                                       4
                                       0
                                      23
                                     47.5
                                      76
                                      84
                                     89.5
GHB119, Cry2Ae
                                      6.9
                                      23
                                      79
                                      100

                                       4
                                     49.5
                                      83
                                     97.5
                                      100

TwinLink(TM)
                                      6.9
                                     27.5
                                      75
                                      100

                                       4
                                      29
                                      80
                                     97.5
                                      100

Modified from MRID 476400-14

   2. Tobacco Budworm

As for TBW, two types of assays were carried out, the diet bioassay and overlay bioassay using all three toxins. The same methodology was used for TBW as was used for CBW (see above Section III c) 1.). However, concentrations for the negative control of diet bioassays differed and were 0, 0.8, and 4% of Coker312. Furthermore, mortality was scored at day 4, 7, 12, and 17 and averaged across the two replicates. For overlay assays, mortality was scored at day 3, 7, 11, and 15.

As for CBW bioassays, control mortality for TBW was high and difficult to control. The applicant attributed this to the presence of gossypol in the leaf diet. BCS reported that control mortality was lower in overlay assays and that therefore those results were best for verifying TwinLink(TM) cotton's dose profile. BCS concludes from the bioassay results using method (1) that there was a high dose expression for TBW in TwinLink(TM) cotton and its individual traits.

Table D.5. TBW mortality to Cry1Ab, Cry2Ae, and TwinLink(TM) - diet incorporated assays
Treatment
                           % Treatment Concentration
                                  % Mortality

                                     Day 4
                                     Day 7
                                    Day 12
                                    Day 17
Food*
                                       0
                                       0
                                      8.3
                                      25
                                      25
Coker312
                                       4
                                       0
                                     14.8
                                     23.8
                                     23.8

                                      0.8
                                       2
                                      17
                                      28
                                      28
T304-40, Cry1Ab
                                       4
                                      29
                                      73
                                     95.5
                                      100
GHB119, Cry2Ae
                                       4
                                      100

TwinLink(TM)
                                       4
                                      100

Modified from MRID 476400-14
* Unclear what source of food constituted negative treatment.

Table D.6. TBW mortality to Cry1Ab, Cry2Ae, and TwinLink(TM) - overlay assay method
Treatment
                           % Treatment Concentration
                                  % Mortality

                                     Day 3
                                     Day 7
                                    Day 11
                                    Day 15
Coker312
                                       4
                                       0
                                     14.8
                                      17
                                      17
T304-40, Cry1Ab
                                       4
                                      41
                                     88.5
                                     95.5
                                      100
GHB119, Cry2Ae
                                       4
                                      76
                                      100

TwinLink(TM)
                                       4
                                      71
                                      100

Modified from MRID 476400-14

High-Dose Verification Method (4):

   1. Cotton Bollworm

Coker312, TwinLink(TM), and its two single events were evaluated for efficacy against CBW and PBW. The experiment was carried out at 14 different locations across the US with two replicates each during the 2007 and 2008 cotton growing season. At two locations only, BCS conducted a larval survival experiment which provided the data for the reported results of high-dose verification method (4).

In two locations (in 2007) and three locations (in 2008) of the Cotton Belt (actual locations were not reported by the applicant), natural infestation was supplemented with artificial infestations of the target pest; repeated artificial infestations with CBW were conducted at one location in 2007 and three locations in 2008. A total of 7,200 eggs were applied in each row of 60 plants and 50 plants in 2007 and were analyzed for presence of larvae. In 2008, a total of 6,600 eggs/plot were applied and each boll was dissected subsequently to determine presence of larvae. Percent mortality was calculated for 2007 (Table D.7). The average number of boll examined in each plot was recorded and percent mortality calculated in 2008 (Table D.8).

BCS reported that mortality for all three events (single and dual) was very high and greater than 99.2% in all but one instance. In 2008, one TwinLink(TM) plot had slightly less CBW mortality (98.5%).

Table D.7. CBW mortality on artificially infested plots during the 2007 growing season
Event
                               Total eggs/plot*
                               Total live larvae
                                  % Mortality
T304-40, Cry1Ab
                                     6000
                                      16
                                     99.73
GHB119, Cry2Ae
                                     6000
                                       1
                                     99.98
TwinLink(TM)
                                     6000
                                       2
                                     99.97
* 50 plants/60-plant row were scouted = 83.3%
Modified from MRID 476400-14

Table D.8. CBW mortality on artificially infested plots during the 2008 growing season
Event
                                  Total eggs/
                                 3 locations*
                                  Total fruit
                                   (average)
                                  Total live
                                    larvae*
                                 % live larvae
                              Est. live larvae**
                                  % Mortality
T304-40, Cry1Ab
                                     19800
                                     30422
                                       8
                                     0.84
                                    322.97
                                     99.52
GHB119, Cry2Ae
                                     19800
                                     24996
                                       3
                                     0.27
                                     86.40
                                     99.68
TwinLink(TM)
                                     19800
                                     32279
                                       7
                                     0.89
                                    309.01
                                     98.26
* 6,600 eggs were applied per plot; 1 plot = 1 location
Modified from MRID 476400-14

   2. Pink Bollworm

A similar artificial infestation study as for CBW was carried out for PBW by Drs. Ellsworth and Li at the University of Arizona. Four plots were planted as a random complete block design (8 replicates) with TwinLink(TM), each single event, and Coker312 as control. Each plot was six rows long (30-ft) and separated from other blocks by one empty row. Young bolls were infested with 100 eggs each from a susceptible PBW strain maintained by USDA-ARS (Maricopa, AZ). Six to eight days later tagged bolls were brought into the lab for dissection. Attack rates on bolls were evaluated by counting the number of entry holes on the exterior of the boll carpel. PBW survival was estimated 6-15 days later which provided enough time for larvae to develop to 3[rd] instar. Survival was determined based on the number of exit holes, 3[rd], live 4[th], and pupae. Mortality (synonymously used with efficacy) was estimated by dividing the number of surviving PBW by the number of entry holes and by correcting for control mortality. Mortality in the Coker line was determined by limiting examinations to mainly those bolls with < 20 entry holes in order to avoid density-dependent mortality in control treatments.

BCS concluded that all three infestations with Cry1Ab and TwinLink(TM) demonstrated high efficacy (corrected mortality >99.5%) against PBW. Two infestations with Cry2Ae demonstrated high efficacy (corrected mortality >99.1%), while results from one infestation were somewhat lower (corrected mortality 98.7%). BCS hypothesized that the small sample size from the first infestation in addition to the high control mortality may have contributed to this lower mortality estimate. Since control mortalities were high (Table D.9), corrected mortality rates based on each infestation showed somewhat lower levels of efficacy for each line. Cry2Ae was somewhat less effective in all infestations. When all data were pooled for each cotton line, the mortality rates were again very high (Table D.10).

Table D.9. Mortality results of susceptible PBW following artificial infestation of cotton plants
Infestation/ Data collection
                                   Treatment
                                  Entry Holes
                               Live Large Larvae
                             Average Mortality (%)
                                   Lower 95%
                                     Mean
                                Upper 95% Mean
                            Corrected Mortality (%)
July
                                     Coker
                                    Cry1Ab
                                    Cry2Ae
                                  TwinLink(TM)
                                     1646
                                     2756
                                     1901
                                      331
                                      726
                                       8
                                      11
                                       2
                                     50.26
                                     99.70
                                     99.23
                                     99.96
                                     46.37
                                     99.46
                                     98.65
                                     99.87
                                     54.16
                                     99.94
                                     99.82
                                      100
                                       -
                                     99.34
                                     98.69
                                     99.86
August
                                     Coker
                                    Cry1Ab
                                    Cry2Ae
                                  TwinLink(TM)
                                     1262
                                     2725
                                     2485
                                     1897
                                      393
                                       0
                                       5
                                       0
                                     59.75
                                      100
                                     99.84
                                      100
                                     54.39
                                      100
                                     99.70
                                      100
                                     65.10
                                      100
                                     99.98
                                      100
                                       -
                                      100
                                     99.35
                                      100
September
                                     Coker
                                    Cry1Ab
                                    Cry2Ae
                                  TwinLink(TM)
                                      598
                                     2399
                                     1551
                                     1452
                                      182
                                       3
                                       4
                                       0
                                     63.29
                                     99.75
                                     99.65
                                      100
                                     56.63
                                     99.32
                                     99.19
                                      100
                                     69.95
                                      100
                                      100
                                      100
                                       -
                                     99.59
                                     99.15
                                      100
 Modified from MRID 476400-14

Table D.10. Pooled mortality results of susceptible PBW following artificial infestation of cotton plants
Treatment
                                 Total Entries
                                    N Bolls
                               Live Large Larvae
                              Apparent Mortality
                                Lower 95% mean
                                Upper 95% Mean
                              Corrected Mortality
                                      (%)
Coker
                                     3506
                                      259
                                     1301
                                     56.3
                                     53.4
                                     59.2
                                      - -
Cry1Ab
                                     7880
                                      345
                                      11
                                     99.8
                                     99.6
                                     99.9
                                     99.6
Cry2Ae
                                     5937
                                      252
                                      20
                                     99.6
                                     99.4
                                     99.8
                                     99.1
TwinLink(TM)
                                     6680
                                      302
                                       2
                                     99.9
                                     99.9
                                      100
                                     99.9
Modified from MRID 476400-14

High-Dose Verification Method #3:

   1. Pink Bollworm

In 2007 and 2008, an artificial infestation study was carried out by Drs. Ellsworth and Li at the University of Arizona with a homozygous resistant strain of PBW. A Cry1Ac-resistant PBW strain (AZP-R) was obtained from Dr. Bruce Tabashnik at the University of Arizona. That strain was resistant to Cry1Ac with 53% survival in response to 10ug of Cry1Ac powder/g of diet and demonstrated cross-resistance to Cry1Aa and Cry1Ab. The artificial infestation experiment was run in the lab with field collected plant tissue of Cockerline (control), TwinLink(TM), Cry1Ab, and Cry2Ae plant tissue.

Young cotton bolls were harvested (15-19 days old) after tagging. Each boll was maintained in a water pick bioassay system. To measure the efficacy of the transgenic cotton, bolls were infested with an estimated 15 homozygous PBW neonates. The same procedures were followed for attack rate estimates, boll dissection, and PBW survival and mortality were the same as described for the PBW artificial infestation study using verification method #4.

A total of 348 cotton bolls were assayed. The mean entry holes ranged from 6.2-28.5. Attack rates were much higher in some cases. Maximum number of entry holes on a boll were 31 (Coker), 48 (Cry1Ab), 48 (Cry2Ae), and 54 (TwinLink(TM)). The mean percentage of PBW recovered ranged from 80.1 to 94.5%, which was similar to assay results with susceptible PBW. The criterion for `survival' at the time of boll dissection was a `living >3[rd] instar larva'. Mortality was calculated by dividing the number of surviving PBW by the number of entry holes as well as by correcting for control mortality. JMP IN Statistical Discovery Software, Version 5.1, SAS Institute 2005 was used for the data analyses.

In two out of three infestations, Cry2Ae demonstrated 100% efficacy; TwinLink(TM) demonstrated 100% efficacy in all three infestations. Cry1Ab treatments were less effective in the first and second infestation and demonstrated slightly better efficacy in the third infestation. However, corrected mortalities for Cry1Ab were similar and just below 50%. When all data were pooled for each treatment (or cotton line), the mortality rates were high. PBW mortalities in Cry2Ae and TwinLink(TM) were not significantly different from 100%. The reported cross-resistance of AZP-R to Cry1Ab toxin was consistent with the findings in this study. No reported cross-resistance to Cry2Ae was apparent. The authors concluded that Cry2Ae and TwinLink(TM) expressed a high-dose against PBW.

The authors stated that PBW survival on Bt plants (TwinLink(TM) and Cry2Ae) could have been higher because 1[st] instar larvae might have been exposed to hemizygous rather than purely homozygous seed tissue. Experimental Bt plot sizes where small, and Coker null lines were in close proximity, which could have facilitated low rates of out-crossing. Also, other factors like environment or altered production of protein due to warting (after bollworm attack) may have led to lower Bt expression in bolls than expected and hence, led to higher survival of PBW on TwinLink(TM) and Cry2Ae plants.

Table D.11. Mortality results for Cry1Ac resistant PBW following artificial infestation of cotton plants
Infestation/ Data collection
                                   Treatment
                                  Entry Holes
                               Live Large Larvae
                            Apparent Mortality (%)
                                   Lower 95%
                                     Mean
                                Upper 95% Mean
                            Corrected Mortality (%)
July
                                     Coker
                                    Cry1Ab
                                    Cry2Ae
                                  TwinLink(TM)
                                      210
                                      466
                                      448
                                      569
                                      86
                                      99
                                       1
                                       0
                                     51.9
                                     76.8
                                     99.8
                                      100
                                     36.0
                                     71.3
                                     99.4
                                      100
                                     67.7
                                     82.3
                                      100
                                      100
                                       -
                                     48.1
                                     99.5
                                      100
August
                                     Coker
                                    Cry1Ab
                                    Cry2Ae
                                  TwinLink(TM)
                                      279
                                      431
                                      467
                                      468
                                      89
                                      71
                                       0
                                       0
                                     67.1
                                     79.8
                                      100
                                      100
                                     60.5
                                     73.1
                                      100
                                      100
                                     73.7
                                     86.5
                                      100
                                      100
                                       -
                                     48.4
                                      100
                                      100
September
                                     Coker
                                    Cry1Ab
                                    Cry2Ae
                                  TwinLink(TM)
                                      111
                                      71
                                      141
                                      327
                                      30
                                       9
                                       0
                                       0
                                     67.5
                                     90.3
                                      100
                                      100
                                     52.6
                                     78.6
                                      100
                                      100
                                     82.4
                                      100
                                      100
                                      100
                                       -
                                     53.1
                                      100
                                      100
Modified from MRID 476400-14

Table D.1. Pooled mortality results for Cry1Ac resistant PBW following artificial infestation of cotton plants
Treatment
                                 Total Entries
                                    N Bolls
                               Live Large Larvae
                              Apparent Mortality
                                Lower 95% mean
                                Upper 95% Mean
                              Corrected Mortality
                                      (%)
Coker
                                      600
                                      56
                                      205
                                     64.5
                                     58.3
                                     70.7
                                       -
Cry1Ab
                                      968
                                      74
                                      179
                                     80.3
                                     76.0
                                     85.6
                                     45.9
Cry2Ae
                                     1056
                                      85
                                       1
                                     99.95
                                     99.85
                                      100
                                     99.7
TwinLink(TM)
                                     1364
                                      112
                                       0
                                      100
                                      100
                                      100
                                      100
Modified from MRID 476400-14

IV.       CROSS-RESISTANCE POTENTIAL (MRID 476400-14)

BCS referred to a series of binding studies (Karim et al., 2000; Luo et al., 2007; Hernandez-Rodriguez et al., 2008) to support that Cry1 and Cry2 proteins bind to different target sites in the larval midgut of Lepidoptera pests. In addition, work by Hernandez-Rodriguez et al. (in press) showed that Cry2Aa, Cry2Ab, and Cry2Ae share a common binding site in CBW and the Old World bollworm.

BCS commissioned Dr. Jurat-Fuentes at the University of Tennessee to conduct competitive binding experiments with radio labeled Cry1Ab and Cry2Ae proteins using BBMV of TBW and CBW to confirm that these proteins bind to the target pests' midgut with specificity. Furthermore, Dr. Jurat-Fuentes conducted a study with Cry1F, Cry1Ab, Cry1Ac, Cry2Ab, and Cry2Ae to fully characterize competitive binding aspects between the two newly proposed Bt toxins for cotton and those toxins already found in already registered Bt cotton products (see figures and results below).

BCS did not conduct binding studies for the third main target pest, PBW; rather the applicant cited publicly available literature. In experiments by Karim et al. (2000), Cry1Ab and Cry1Ac bound specifically and with high affinity to BBMV of PBW, while Cry1Aa and Cry2Aa bound with less specificity and lower affinity yet showed high binding site concentration. BCS stated that these results confirmed that Cry1A and Cry2A proteins were unlikely to be for cross-resistant, as was seen in other insect studies.

   1. Binding Experiments with Cry1Ab and Cry2Ae using CBW Brush Border Membrane Vesicles

The BBMV experimental results showed that labeled Cry1Ab (see Figure 2) and Cry2Ae (not shown here) bound with specificity to the midgut of CBW.

Figure D.2. BBMV binding assays with increasing CBW  BBMV concentrations and fixed radio-labeled Cry1Ab (from MRID476400-14). Unlabeled and labeled toxin (of constant amount) tend to bind to specific sites first; once the specific receptors are occupied, toxins bind to unspecific sites. Non-specific binding continues to increase linearly because the concentration of non-specific sites increases with increasing BBMV.

   2. Binding Experiments with Cry1Ab and Cry2Ae using TBW Brush Border Membrane Vesicles

The BBMV experimental results showed that labeled Cry1Ab (see Figure 3) bound with specificity to the midgut of TBW. The results for Cry2Ae are not shown here.

Figure D.3. BBMV binding assay with increasing TBW BBMV concentrations and fixed radio-labeled Cry1Ab (from MRID476400-14)

   3. Competition Experiments between Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and Cry2Ae using CBW Brush Border Membrane Vesicle

A competitive binding experiment with Cry1Ab, Cry1Ac, Cry1F, Cry2Ab2, and Cry2Ae showed that Cry1Ab shared high affinity binding sites with Cry1Ac and Cry1F in CBW. However, Cry1Ab did not compete with Cry2Ab and Cry2Ae suggesting that these proteins do not share binding sites in CBW (See Figure 4).

Figure D.4. Competition binding of Cry1Ab and competitors to BBMV of CBW (from MRID 476400-14); if two different Bt toxins share binding sites, there will be less binding by the labeled toxin (with constant concentration) as the amount of competitor increases. When there is no competition, a flat line across can be expected for the competitor concentration.

   4. Competition Experiments between Cry1Ab and Cry2Ae using TBW Brush Border Membrane Vesicles

Results of a binding competition experiment between Cry1Ab and Cry2Ae using TBW BBMV showed that the two toxins did not share binding sites (Figure 5).

Figure D.5. Competition assay with Cry1Ab and Cry2Ae using BBMV of TBW

In conclusion, BCS stated that the relative potential for cross resistance was low between variants of Cry1 and Cry2 toxins as expressed in Bt cotton. Yet, the potential for cross-resistance was high within the group of Cry1 variants (e.g. Cry1Ab, Cry1Ac, and Cry1F) as well as within the group of Cry2 variants (e.g. Cry2Ab and Cry2Ae) (see Table below).

Table D.2. BCS' estimated potential for cross-resistance between various Bt proteins in TBW, CBW, and PBW
                                     Toxin
                                    Cry1Ab
                                    Cry1Ac
                                     Cry1F
                                    Cry2Ab
                                    Cry2Ae
Cry1Ab
                                      N/A
                                      +++
                                      ++
                                       -
                                       -
Cry1Ac

                                      N/A
                                       +
                                       -
                                       -
Cry1F

                                      N/A
                                       -
                                       -
Cry2Ab

                                      N/A
                                      +++
      Modified from MRID 476400-14
      +++, ++ = high potential; + = low potential; - = little to no potential

V.       MODELING

BCS commissioned Dr. Mike Caprio (Mississippi State University) to conduct a risk assessment for TwinLink(TM) cotton on CBW using a model by Caprio et al. (2009). Dr. Caprio conducted a probabilistic risk assessment using a deterministic model and using PERT distributions (type of Beta distribution with mean was weighted, for example, 4x more than minimum and maximum values) for parameters of interest (with the exception of two cases: dominance associated with fitness costs). One thousand simulations were run with reasonable parameter values chosen at random from distributions derived with expert feedback (Table D.14).

The model included 43 different habitats from early season wild hosts, non-Bt corn, Bt cotton, soybeans, sorghum, and late season wild hosts. A field of cotton progressed through four different habitats in a growing season: fallow field (unsuitable habitat), pre-bloom cotton, flowering cotton, peak bloom cotton, and back to fallow field. Two Bt traits were modeled, Cry1Ab was assumed to have complete cross-resistance to Cry1Ac, and Cry2Ae was assumed to have complete cross-resistance to Cry2Ab2.

The size of each field was assumed to be 100 acres, and the modeling landscape consisted of 1024 fields. Corn, cotton, and soybeans accounted for 260 fields each, sorghum for 99 fields, and wild hosts for 145 fields. 100% of cotton was simulated as TwinLink(TM) cotton. No Cry1Ab corn was modeled in the landscape because BCS did not have access to sales information.

The rate of resistance evolution was estimated in two ways: (1) The final R-allele frequency after 31 years was divided by the initial frequency (was not constant due to re-sampling) and the result was log transformed. In many cases, the resistance allele frequency exceeded 90% and, once there, could no longer increase. All cases that approach the frequency of 1.0 received the same score no matter how many years it took to approach that value. (2) The rate of change in the resistance allele frequency was measured after three years to estimate the rate of resistance evolution.

Table D.3. Parameters varied and used to define probability function, and function used for random draws
Parameter
                                      Max
                                      Min
                                  Most Likely
                                 Distribution
Initial R-allele frequency, Cry2Ae
                                     0.01
                                    0.00001
                                    0.0002
                                   Pert-Log
Initial R-allele frequency, Cry1Ab
                                     0.05
                                    0.00001
                                     0.002
                                   Pert-Log
R-allele fitness cost (independently drawn for both alleles)
                                      0.5
                                      0.0
                                      0.1
                                     Pert
R-allele fitness cost dominance (independently drawn for both alleles)
                                      0.2
                                      0.0
                                     0.05
                                  Mpert (2.0)
Mortality from Cry2Ae
                                    0.9999
                                     0.999
                                     0.99
                                   Pert-Log
Mortality from Cry1Ab
                                    0.99999
                                    0.9999
                                     0.99
                                   Pert-Log
Dominance of resistance (independently drawn for both alleles)
                                      0.3
                                     0.00
                                     0.05
                                     Pert
Modified from MRID 476400-14

In the model, resistance did not evolve to either toxin in TwinLink(TM) cotton in 1000 simulations of 31 years of use; hence, under the conditions modeled there appeared little risk of product failure in a 30-year time horizon. In none of the simulations did the resistance allele frequency increase more than three-fold (see Figures 6 and 7); in most cases the R-allele frequency decreased. The fitness costs simulated were low (recessive in nature), however, the effects of fitness costs exceeded the effects of selection. The report was concluded with "These results support the contention that even small fitness costs can have a much larger impact on the evolution of resistance to stacked toxins than the case of plants expressing a single toxin".

Figure D.6. Rate of increase in Cry1Ab resistance allele frequency after three years. Decreases in the resistance allele frequency occur when fitness costs exceeded increases due to selection (taken out of MRID 476400-14)

Figure D.7. Rate of increase in Cry2Ae resistance allele frequency after 3 years

VI.       REFUGE STRATEGY

BCS stated that since 1) both Cry1Ab and Cry2Ae express high-dose for the major target pests, CBW and TBW, and 2) the two toxins bind to different target sites in the midgut of the three insect pests, that TwinLink(TM) cotton should qualify for Natural Refuge. Their request was accompanied by a letter from Monsanto Company which gave BCS permission to cite relevant studies in support of a natural refuge request.

A waiver request for an IRM plan was submitted to the Agency because BCS does not intend to commercially sell seeds from the single events Cry1Ab and Cry2Ae. Very small acreages may be grown for seed production only in order to test combinations with other insect control traits that could potentially be candidate commercial products.

BCS submitted a structured refuge request for the target pest pink bollworm.

VII.       RESISTANCE MONITORING PROGRAM

BCS proposed to monitor for resistance evolution in all three target pests in areas of high TwinLink(TM) adoption.

The applicant proposed to have baseline susceptibility established for Cry1Ab and Cry2Ae proteins by 2012 (for CBW, TBW, and PBW) prior to commercialization. Sample collection for TBW and CBW would occur in the following manner: 10-20 populations for each pest would be collected in the primary cotton growing regions of the USA. For PBW, 4-6 populations would be collected from Arizona, New Mexico, West Texas, and California. This preliminary susceptibility data will provide valuable baseline information to compare with future annual insect susceptibility monitoring data. BCS will also use this baseline information to develop a discriminating dose for each toxin and each pest.

Once TwinLink(TM) has been commercialized, BCS has committed to monitoring of the three target pests on an annual basis.

VIII.       REMEDIAL ACTION PLAN

BCS stated that if insect resistance is suspected (monitoring or unexpected crop damage reports), it will attempt to confirm and characterize the resistance. First, the damaged cotton plants would be tested to confirm that the Bt proteins was produced and that a target pest of TwinLink(TM) cotton was responsible for the observed damage. If the plants were confirmed to express TwinLink(TM), the insect damage was from a primary target pest, and all other reasonable causes for the field damage had been ruled out (i.e. weather, cultural reasons), then insect populations would be collected from the region. Protein and plant bioassays would be conducted to determine the level and extent of resistance on the sampled insect populations and a colony established to determine if the putative resistance was heritable. Data from standardized bioassays would be compared to the original baseline data. Communication with customers and extension agents in the region, the EPA, and other registrants and applicants, whose products could be impacted by the findings, is another aspect of BCS' proposed remedial program.

Confirmed resistance would define the remedial action area(s) as well as the approach. The main focus would be to limit the spread of resistant alleles by eliminating resistant population(s) as quickly as possible. This might be accomplished through chemical insecticide treatments of the Bt cotton field effective against the resistant insects and/or using crop destruction methods that could limit the growth and reproduction of the resistant population(s). Susceptibility monitoring would be enhanced, especially in the region where resistance was identified. Refuge acreage may be modified and/or suspension of product sales could occur in the affected areas, not only TwinLink(TM), but for all products expressing similar Bt proteins that could be potentially cross-resistant.

IX.       GROWER EDUCATION

BCS stated that it will use grower meetings to introduce the TwinLink(TM) technology and guide the growers on best practices to capture the highest production yields. Growers have the responsibility to monitor for unexpected crop damage in TwinLink(TM) and to report product failure to BCS. A toll free telephone number will be provided in brochures and other communication tools for efficient tracking and follow-up of unexpected insect damage reports.

BCS will ensure that IRM plans are fully communicated to BCS sales staff and distribution channels and initially, will limit TwinLink(TM) seed sales to its own seed company, under FiberMax and Stoneville varieties. This will provide BCS with oversight on communication and control with educational efforts of sales staff. The transition to a natural refuge for combined trait Bt cotton products will reduce the education/communication needs related to refuge deployment and eliminate the need to monitor grower compliance with IRM requirements aside from the required field monitoring for unexpected insect damage.

X.       GROWER COMPLIANCE

Grower compliance with refuge and IRM requirements is a critical element for resistance management. Significant non-compliance with IRM among growers may increase the risk of resistance for Bt crops. To minimize the effects of non-compliance, it is necessary to develop a broad compliance program as part of the IRM strategy. Such a program has to include 1) an understanding of the effect of non-compliance on IRM; 2) identification of compliance mechanisms to maximize adoption of IRM requirements; 3) measurement of the level of compliance; and 4) establishment of an enforcement structure to ensure compliance and penalize non-compliance.

XI.       REPORTING REQUIREMENTS

Once the baseline susceptibility data is collected for the three primary target pests and a discriminating dose level has been established, annual monitoring will be conducted and reported to the agency.

XII.       BPPD REVIEW

A. PROTEIN EXPRESSION

BPPD reviewed BCS' protein expression results reported in their IRM submission and concludes that single event T304-40 may not express the same levels of toxin as in the dual gene PIP, TwinLink(TM). It appears that T304-40 was expressed at higher levels in leaves of single event plants compared to leaves collected from TwinLink(TM) plants. For flowers and bolls the pattern was reversed, and TwinLink(TM) plants appeared to express T304-40 at higher levels in these tissues. It is unclear whether these differences in expression results were statistically significant because BCS did not provide further information on the differences observed.

Expression of GHB119 in leaves from early to late stage appeared somewhat variable or unstable. Expression levels were measured in leaves in 2004 and 2005 at three developmental stages (early leaf, mid leaf, and late leaf). At developmental stage 1, expression was higher than at developmental stage 2, and at developmental stage 3, expression increased again but to higher levels than observed at developmental stage 1. For early, mid, and late boll, Expression level for GHB119 were also assessed in three different developmental stages of bolls (early, mid, and late). Levels of GHB119 were higher in TwinLink(TM) than in the single event plant during developmental stage 2 and 3. It appears that the difference was approximately two-fold (see Figure D.1).

Table D.1 in this review summarizes the protein expression level (ug/g-dry weight) submitted by BCS for T304-40 and GHB119 in different plant tissues. It appears that the same tissue types were lumped together for plants expressing single and pyramided events. Since there appears to be unequal expression of the proteins in plants expressing either the single or pyramided events over the course of the growing season, this approach is not providing the most information. It would be more useful to have the expression data broken down by tissue for each individual event, GHB119, T304-40, and TwinLink(TM), rather than lumped together for the same protein.

BPPD recommends that BCS provide more specific and explanatory information together with their protein expression results in future data submissions for PIP registrations.

B. DOSE

The determination of dose, or the amount of toxin expressed by the transgenic crop relative to the susceptibility of the target pests, is a critical component of IRM. Models have shown that a high-dose of toxin, coupled with a non-transgenic refuge to provide a supply of susceptible insects, is the most effective strategy for delaying resistance in Bt crops. The high-dose/refuge strategy assumes that resistance to Bt is recessive and is conferred by a single locus with two alleles resulting in three genotypes: susceptible homozygotes (SS), heterozygotes (RS), and resistant homozygotes (RR). The high-dose/refuge strategy also assumes that there will be a low initial resistance allele frequency and extensive random mating between resistant and susceptible adults. In practice, a high-dose PIP should express sufficient quantities of toxin to kill all susceptible insects (SS) as well as heterozygous insects with one resistance allele (RS). Lower dose PIPs might allow for survival of insects with at least one susceptibility allele (SS or RS), although effective IRM may still be possible with a suitable refuge strategy. To be able to demonstrate high-dose, it is recommended that applicants generate data by at least two of the five laboratory and field approaches as outlined by the SAP (1998) and described by the Agency in the 1998 Bt Plant-Pesticides and Resistance Management document (US EPA, 1998) and 2001 Biopesticide Registration Action document (US EPA, 2001).

The high-dose definition and verification techniques were developed in 1998 when all of the registered Bt crops were single toxin products targeted against Lepidopteran pests. In recent years, PIPs in Bt cotton have been approved that contain two genes targeted at the same insect pest. These "pyramided" products can be beneficial for IRM since target pests must overcome two toxins to develop field resistance to the PIP. The benefits are greatest for two toxins with unrelated modes of action (i.e. binding to different Bt receptor sites in the midgut) that are expressed at high-doses in the plant (Roush 1998).

For pyramided products, the dose of each toxin should be evaluated separately. In cases where the pyramided product is created through conventional breeding and the dose of the single toxin products has already been established, the combined dose in the pyramided PIP can be determined with comparative efficacy studies and protein expression data. However, this is not the case for TwinLink(TM) cotton.

BPPD reviewed BCS' dose information for TBW, CBW, and PBW and concludes the following:

   1. Tobacco Bollworm

BCS submitted one dose verification method for TBW rather than two. Since all verification methods may be imperfect at assessing dose against a target pest when used alone, the Scientific Advisory Panel (1998) recommended that at least two methods (of five methods described by the panel) be used when demonstrating dose expression in target pests. BPPD notes that a second dose verification method is lacking for TBW. In the past Natural Refuge requests were granted on the basis that the toxins expressed high-dose against TBW. A second dose-verification method will need to be submitted if BCS wishes to maintain that GHB119, T304-40, and TwinLink(TM) express a high-dose against TBW and to support their Natural Refuge request.

High-dose verification method (1):
From mortality results obtained with dose verification method (1), BPPD concludes that events GHB119, T304-40, and TwinLink(TM) may express a high-dose and effective high-dose, respectively, against TBW.

After day 4 of the diet-incorporated bioassay, mortality was reported as 100% for TwinLink(TM) and Cry2Ae treatments, while control mortality ranged from 0%-2%. For the overlay assay, mortality was 100% for TwinLink(TM) and Cry2Ae by day 7, while control mortality was 15%.

For Cry1Ab treatments, mortality reported from diet bioassays did not reach 100% until after day 15. In addition, control mortality was relatively high (ranging from 23%-28%). For overlay assays, mortality for TBW when exposed to Cry1Ab was 100% by day 15; control mortality at that time had reached 17%. Cry1Ab may express a high-dose against TBW.

   2. Cotton Bollworm

It appears that TwinLink(TM) may express a possible effective high-dose against CBW. However, a second dose verification method turned out to be inconclusive during BPPD's review and therefore, a high-dose cannot be verified. Should BCS wish to maintain that TwinLink(TM) expresses a high-dose against CBW, additional information needs to be provided to clarify uncertainties in the experiment using high-dose verification method 4.

High-dose verification method (1):
BPPD concludes that the diet-incorporated and overlay assays together suggest that TwinLink(TM) and GHB119 may express a high-dose, but T304-40 may express less than high-dose against CBW.

In the control treatments, larvae had developed into pupae by day 17. BCS did not report whether surviving larvae of the exposure treatments, Cry1Ab, Cry2Ae, and TL, developed into pupae and consequently adults or whether survivors remained in the larval stage. BPPD notes that for future submission, it is recommended that BCS report whether surviving larvae were developmentally `dead' (as opposed to physiologically), or whether they continued to mature into pupae and adults.

High-dose verification method (4):
BPPD identified several uncertainties in the methodology section that preclude making a dose determination based on study results obtained with dose verification method (4).

BCS stated in the IRM submission that mortality results were obtained from an artificial and natural infestation experiment done in conjunction with the efficacy study described in chapter 13 of their submission (MRID 476400-12). When BPPD consulted chapter 13 for raw data or data tables, however, mortality data from the infestation experiments were lumped for all lepidopteran pests, and no pest specific mortality data were reported. It is unclear how the data that BCS reported in chapter 15 of their submission (Tables 7 and 8) were obtained, and no further details about the study were submitted in an appendix accompanying their submission (MRID 476400-14).

Furthermore, BCS stated in study volume 13 that some plots were treated with conventional pesticides, while others were not. It is unclear whether the mortality results reported in Table 7 and Table 8 come from plots that have been sprayed, not sprayed, or both, and what effect spraying might have had on dose determinations.

Coker cotton was planted in the control plot treatments for this natural and artificial infestation experiment; however, no information on control mortality was provided in the submitted report. It cannot be determined whether the final CBW mortality results from plots expressing Cry1Ab, Cry2Ae, and TwinLink(TM) have been corrected for control mortality.

The applicant may have conducted an egg hatchability study along with the infestation experiment to estimate egg viability and larval attack rate. BPPD has not been provided with egg hatchability data or other information that would allow estimating viability and attack rate. Without such information it is unclear how many larvae hatched, attacked the Bt cotton plants, and ultimately survived Bt exposure. The applicant did, however, provide the infestation rate per plant.

Furthermore, BCS provided BPPD with CBW average mortality across two locations in 2007, and three locations in 2008, but no results were provided for individual locations. In addition to providing average mortality results, BPPD requests that individual treatment mortalities be reported for future data submissions so that minimum mortality per toxin is known.

Should BCS wish to pursue the high-dose claim for TwinLink(TM) and CBW, BPPD recommends providing clarification and further data for the study using high-dose verification method (4).

BPPD further recommends that the applicant provide more in-depth descriptions of results and methodology for future studies and data submissions.

   3. Pink Bollworm

High-dose verification methods (3) and (4):
BPPD cannot make a conclusion about the dose profile for TwinLink(TM) and its events and PBW (using verification methods 3 and 4) because it is unclear how the mortality results reported by Ellsworth and Li (2008) were obtained. When BPPD divided the number of entry holes reported (Tables 9-12) by the number of live larvae and calculated average mortality, the obtained results did not match mortalities reported by BCS.

Deficiency 1: The dose profile reported for PBW cannot be verified by BPPD until BCS clarifies how mortalities were calculated for the experiments using dose verification methods (3) and (4).

Table D.4. BPPD Conclusion Regarding Dose Profile for Events T304-40, GHB119, and TwinLink(TM)
                                     Pest
                                   Method #1
                                   Method #4
                                   Method #3

                                    T304-40
                                    GHB119
                                      TL
                                    T304-40
                                    GHB119
                                      TL
                                    T304-40
                                    GHB119
                                      TL
CBW
                                 Non-High Dose
                              Possible High-Dose
                         Possible Effective High-Dose
                                   Inconcl.*
                                   Inconcl.*
                                   Inconcl.*
                                      --
                                      --
                                      --
TBW
                              Possible High-dose
                                   High-Dose
                              Effective High-Dose
                                      --
                                      --
                                      --
                                      --
                                      --
                                      --
PBW
                                      --
                                      --
                                      --
                                   Inconcl.*
                                   Inconcl.*
                                   Inconcl.*
                                      ---
                                   Inconcl.*
                                   Inconcl.*
* Insufficient information was submitted to the Agency to make a dose determination
** Cry1Ab could not be tested in this experiment because the lab strain was resistant to Cry1Ab.

C. CROSS-RESISTANCE

Analyses of resistance to Bt Cry proteins indicate that cross-resistance occurs most often with proteins that are similar in structure (Tabashnik, 1994; Gould et al., 1995). BCS cited the work by Crickmore et al. (1998), which supports that Cry1 and Cry2 Bt proteins do not have more than 20% amino acid sequence homology in common. Furthermore, the 3-dimensional crystal structures of Cry2Aa (very closely related to Cry2Ae as expressed in TwinLink(TM)) and Cry1Aa (very closely related to Cry1Ab as expressed in TwinLink(TM) proteins have been resolved by Morse et al. (2001) and demonstrate that the proteins have a distinct tertiary structure. This significant difference in structure suggests that these two classes of proteins may also have unique biochemical mechanisms, specifically with respect to binding sites. The similarity in the global architecture of Cry2Aa and Cry1Aa, however, suggests that the two toxins may have similar general steps for the mode of action.

BPPD concludes that the evidence provided by BCS supports that cross-resistance between Cry1Ab and Cry2Ae, as expressed in TwinLink(TM) cotton, is unlikely to develop in the main target pests CBW, TBW, and PBW. To summarize, the amino acid sequence homology between the two Bt toxins is less than 20%; the two protein structures differ substantially at the third terminal suggesting that the two proteins may have unique mechanisms with respect to binding sites (and other biochemical pathways); the binding assay studies by Jurat-Fuentes showed that Cry1Ab and Cry2Ae were not competing for the same binding sites in CBW as well as TBW; and experiments by Karim et al. (2000) demonstrated that Cry1Ab binding to gut membrane of PBW was specific while Cry2A binding was less specific yet with high binding site concentrations (suggesting that toxins were not competing for same binding sites).

However, BPPD notes that should resistance evolve in CBW and TBW to Cry2Ae as expressed in TwinLink(TM) cotton that Cry2Ab2 (as in Bollgard II) may be compromised as well because, as BCS stated in their report, there is a great potential for cross-resistance between the  two toxins.

BPPD notes that the cross-resistance potential is great between Cry1Ab, Cry1Ac, and Cry1F (although somewhat less for Cry1F) based on the competition assays; the three toxins share high affinity binding sites. Should CBW evolve resistance to Cry1Ab, other PIP products expressing Cry1Ac and Cry1F will likely be compromised as well.

D. MODELING

EPA has used predictive models to compare IRM strategies for Bt crops. Because models cannot be validated without actual field resistance, models have limitations and the information gained from the use of models is only a part of the weight of evidence used by EPA in assessing the risks of resistance development. It was the consensus of the SAP (2000) Subpanel that models were an important tool in determining appropriate Bt crop IRM strategies. They agreed that models were "the only scientifically rigorous way to integrate all of the biological information available, and that without these models, the Agency would have little scientific basis for choosing among alternative resistance management options." They also recommended that models must have an agreed upon time frame for resistance protection. For example, conventional growers may desire a maximum planning horizon of five years, while organic growers may desire an indefinite planning horizon. The Subpanel recommended that model design should be peer reviewed and parameters validated. Models should also include such factors as level of Bt crop adoption, level of compliance, economics, fitness costs of resistance, alternate hosts, spatial components, stochasticity, and pest population dynamics.

BPPD reviewed the modeling assessment and accepted the probabilistic approach presented therein. Based on the choice for parameter values, natural refuge assumptions, and their analysis, BCS stated that there is little risk of resistance evolution in CBW to TwinLink(TM) within 30 years. BPPD needs further clarifications on several issues and has some concerns regarding assumptions made for certain important parameters. These are summarized below.

Deficiency 2: In the modeling analysis provided by BCS, only CBW was modeled but not TBW. For a new refuge strategy, such as Natural Refuge, each main target pest needs to be evaluated separately via a simulation model that incorporates biology, genetics, toxicity, and ecology of the target pest. BCS needs to provide simulation modeling for TBW in order to support their natural refuge request.

Deficiency 3: The natural refuge paradigm was approved for Bollgard II and WideStrike with a high-dose profile for TBW. If BCS wishes to pursue the natural refuge strategy for TwinLink(TM), then a second high-dose verification study will need to be submitted to support that the PIP expresses a high-dose against TBW, or BCS can design a model with their specific dose data for TBW.

Deficiency 4: CBW is the pest of most concern since its populations show greater genetic variability to toxins expressed in Bt cotton (Cry1Ac, Cry1F, and Cry2Ab2) than other pest (i.e. TBW). BCS's LC50 data show that CBW have a wide range of susceptibility against Cry1Ab (462 -2844 ng/cm[2]) as expressed in cotton when compared to TBW (4.9-10.5 ng/cm[2]). BCS concluded in their report that TwinLink(TM) expressed less than high-dose against CBW due to the target pest being less susceptible to Cry1Ab and lower level of expression of Cry1Ab in the parental event for TwinLink(TM). In addition, bioassay results for Cry1Ab in corn (BPPD 2009) showed greater variability as well for Corn earworm, CEW, (aka CBW) than for other pests, such as ECB. In fact, there is up to a three-fold difference in magnitude of reported LC50s of CEW compared to ECB. And previous attempts to create a diagnostic concentration for CEW were frustrated by the fact that the insect was less susceptible to Cry1Ab than other Lepidopteran pests. Given this information, BPPD is concerned with the dose assumptions for CBW and recommends that an additional analysis be conducted using somewhat lower mortality assumptions based on the study results reported by BCS. The bioassay experiments showed that Cry1Ab caused mortality ranging 88.5-100% (see this review). Estimated mortalities from the artificial infestation experiments (2007-2008) ranged from 99.5-99.7% (see this review). However, BPPD cannot verify the results of the artificial infestation experiments due to lack of information provided (i.e. % egg viability). At the moment, there is no evidence that Cry1Ab expresses high-dose against CBW; hence BPPD recommends exploring dose mortalities ranging from 90-99% in the simulation model.

Deficiency 5: BPPD is concerned about the most likely value chosen for initial resistance allele frequency for Cry1Ab (0.0002). Although no field resistance to Cry1Ab has been documented yet for corn earworm (CEW), the toxin has been in use for over a decade (in Bt corn) and selection may likely have increased the frequency of potential resistance alleles. However, because corn earworm does not overwinter in the Corn Belt, BPPD agrees that the most likely value will be somewhat lower than could otherwise be expected. BPPD recommends that BCS consider modeling a most likely resistance allele frequency of 0.001 for Cry1Ab in CBW.

Deficiency 6: BCS needs to include Cry1Ab corn and Cry1Ab cotton in the modeling landscape when assessing the risk of resistance evolution in the target pests because Syngenta has registered Bt cotton and Bt corn and Monsanto has registered Bt corn products expressing Cry1Ab. These PIPs are represented in the Cotton Belt and need to be included in a simulation analysis for TwinLink(TM).

E. REFUGE STRATEGY

An IRM plan waiver request for single trait events (Cry1Ab and Cry2Ae) is acceptable if seeds from those events are not sold commercially and serve only for producing the combination PIP TwinLink(TM). For any other combinations, BCS may need to apply for an experimental use permit or section 3 registration. BPPD recommends the use of acreage/geographic limitations for the single trait events as a means to mitigate potential resistance development. BCS should provide information on proposed acreage for these single trait events.

BPPD notes that a second dose verification method is lacking for TBW. In the past Natural Refuge requests were granted on the basis that the toxins expressed high-dose against TBW. A second dose-verification method will need to be submitted to support BCS' Natural Refuge request, or BCS can design a model with their specific dose data for TBW.