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.

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.

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.

If a Natural Refuge strategy for TwinLink(TM) 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.

For those States and regions requiring a non-Bt refuge, the following refuge planting requirements are recommended:

1)  5% External, Unsprayed Refuge

At least 5 acres of non-Bt cotton (refuge cotton) must be planted for every 95 acres of Bt cotton. The size of the refuge must be at least 150 feet wide, but preferably 300 feet wide. This refuge may not be treated with any insecticide labeled for the control of tobacco budworm, cotton bollworm, or pink bollworm. At the pre-squaring cotton stage only, the refuge may be treated with any lepidopteran insecticide to control foliage feeding caterpillars. A non-Bt cotton refuge has to be maintained within at least (1/2)  linear mile (preferably adjacent to or within 1/4  mile or closer) from the Bt cotton fields.

2)  20% External Sprayed Refuge
At least 20 acres of non-Bt cotton are planted as a refuge for every 80 acres of Bt cotton (total of 100A). The non-Bt cotton may be treated with insecticides (excluding foliar Btk products) for control of the tobacco budworm, cotton bollworm, or pink bollworm. A non-Bt cotton refuge has to be maintained within at least 1 linear mile (preferably within (1/2) mile or closer) from the Bt cotton fields.

3)  5% Embedded Refuge

The refuge cotton must be planted as at least one single non-Bt cotton row for every six to ten rows of Bt cotton. The refuge may be treated with any insecticide (excluding foliar Btk products) for the control of pink bollworm whenever the entire field is treated. The in-field refuge rows may not be treated independently of the surrounding Bt cotton field in which it is embedded.

F. RESISTANCE MONITORING

The need for proactive resistance detection and monitoring is critical to the survival of Bt technology. The Agency mandates that applicants monitor for insect resistance (measurement of resistance-conferring alleles) to the Bt toxins as an important early warning sign to developing resistance in the field and whether IRM strategies are working. Grower participation (e.g., reports of unexpected damage) is also important for monitoring. Resistance monitoring is also important because it provides validation of biological parameters used in models. However, resistance detection/monitoring is a difficult and imprecise task. It requires both high sensitivity and accuracy. Good resistance monitoring should have well-established baseline susceptibility data prior to introduction of Bt crops. The chances of finding a resistant larva in a Bt crop depend on the level of pest pressure, the frequency of resistant individuals, the location and number of samples that are collected, and the sensitivity of the detection technique. Therefore, as the frequency of resistant individuals or the number of collected samples increases, the likelihood of locating a resistant individual increases (Roush & Miller 1986). If the phenotypic frequency of resistance is one in 1,000, then more than 3,000 individuals must be sampled to have a 95% probability of one resistant individual (Roush & Miller 1986). Current sampling strategies have a target of 100 to 200 individuals per location. Previous experience with conventional insecticides has shown than once resistant phenotypes are detected at a frequency >10%, control or crop failures are common (Roush & Miller 1986). Because of sampling limitations and monitoring technique sensitivity, resistance could develop to Bt toxins prior to it being easily detected in the field. (http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm)

BCS' approach for developing baseline data for TBW, CBW, and PBW by 2012 may be too protracted in the event 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.

The applicant proposed to monitor for resistance evolution in all three target pests 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.

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 the other Bt cotton registrations.

G. REMEDIAL ACTION PLAN

Remedial action plans are a potential response measure should resistance develop to Bt crops.
Since resistance may develop in "localized" pest populations, it may be possible to contain the resistance outbreak before it becomes widespread. A specific remedial action plan should clearly indicate what actions the applicant will take in cases of "suspected" resistance (i.e., unexpected damage) and "confirmed" resistance. The remedial action plan can also include appropriate adaptations for regional variation and the inclusion of appropriate stakeholders. To fully mitigate resistance, a critical element of any remedial action plan should be that once pest resistance is confirmed, sales of all Bt cotton hybrids that express a similar protein or a protein in which cross-resistance potential has been demonstrated would be ceased in the affected region (see http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm)

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.

BPPD notes that it will not be sufficient if BCS only 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 eventually conclusively determine whether the cause of crop damage is due to resistance or other reasons. BPPD understands that it can take several months before a determination can be reached.

H. GROWER EDUCATION

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. Those regions are Arizona, California, and New Mexico and the following Texas counties:  Brewster, Crane, Crockett, Culberson, El Paso, Hudspeth, Jeff Davis, Loving, Pecos, Presidio, Reeves, Terrell, Val Verde, Ward, and Winkler.

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

I. GROWER COMPLIANCE

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

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. These states and regions are 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.

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.

J. REPORTING REQUIREMENTS

BCS stated that "once the baseline susceptibility data were 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:

   * Baseline and discriminating dose work irrespective of their status;
   * Sales data;
   * Annual resistance monitoring; and
   * CAP results for cotton growing regions where Natural Refuge is not applicable.
REFERENCES

Bushey, D. 2008. Request for a waiver from the requirement to develop an insect resistance management plan. Unpublished report submitted by BCS to the Agency, MRID 47634823

Caprio, M.A., Parker, C.D. and J.C. Schneider. Est. 2009. Future fitness of female insect pests in
temporally stable and unstable habitats and its impact on habitat utility as refuges for insect
resistance management. J. Insect Sci. in press

Crickmore, N., Zeigler, D.R., Feitelson, J., Schnepf, E, Van Rie, D., Lereclus, D., Baum, J., and Dean, D.H. 1998. Revisions of the Nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, Vol. 62 (3): 807-813.

Gould, F., Anderson, A., Reynolds, A., Bumgarner, L., and Moar, W. 1995. Selection and genetic analysis of a Heliothis virescens (Lepidoptera: Noctuidae) strain with high levels of resistance to Bacillus thuringiensis toxins. J. Econ. Entomol. 88:1545-1559.

Hernández-Rodriguez, C.S., Van Vliet, A., Bautsoens, N., Van Rie, J. and J. Ferré (est. 2008) Specific binding of Bacillus thuringiensis Cry2A insecticidal proteins to a common site in the midgut of Helicoverpa sp. Appl. Environ. Microbiol. In press.

Karim, S., Riazuddin, S., Gould, F., and D.H. Dean. 2000. Determination of receptor binding properties of Bacillus thuringiensis δ-endotoxins to cotton bollworm (Heliocoverpa zea) and pink bollworm (Pectinophora gossypiella) midgut brush border membrane vesicles. Pest. Biochem. Physiol. 67:198-216.

Luo S., Wu K., Tian Y., Liang G., Feng X., Zhang J., and Y. Guo. 2007. Cross-resistance studies of Cry1Ac-resistant strains of Helicoverpa armigera (Lepidoptera: Noctuidae) to Cry2Ab. J. Econ. Entomol. 100:909-915.

MacIntosh, S., Holloway, J., Jansens, S., and Van Rie, J. 2008. Insect resistance management plan: TwinLink(TM) Cotton. Unpublished report submitted by Bayer CropScience to the Agency, MRID 47640014.

Roush, R. T., and G. L. Miller. 1986. Considerations for design of insecticide resistance monitoring programs. Journal of Economic Entomology, Vol. 79: 293-298.

Roush, R. 1996. Two-toxin strategies for management of insecticidal transgenic crops: can pyramiding succeed where pesticides mixtures have not? Phil. Trans. Royal Soc. Lond. B (1998): 1777- 1786

Tabashnik, B.E. 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol. 39:47-79.

U.S. EPA, 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and Resistance Management, February 9 and 10, 1998

U.S. EPA, 2000. FIFRA Scientific Advisory Panel Subpanel on Bt Plant-Pesticides Risk and Benefit Assessments: Insect Resistance Management, October 18-20, 2000

U.S. EPA, 2001. Biopesticides Registration Action Document  -  Bacillus thuringiensis Plant Incorporated Protectants, http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

BPPD. 2009. Review of 2007 monitoring data submitted by ABSTC for lepidopteran Bt corn registrants (MRID 47413901). Memorandum from S. Borges to M. Mendelsohn dated April 8[th], 2009.

Appendix D. II - EPA's Review of BCS' Response to Deficiencies

Below is EPA's review of BCS' response to the eight IRM deficiencies noted in BPPD's risk assessment for TwinLink(TM) dated 4/22/2010 (EPA Reg. Nos. 264-RNOU, 264-RNOL, and 264-RNOA, MRIDs 4760014 and 47634823). On July 29[th], 2010, BCS submitted their response to BPPD's eight listed IRM deficiencies for TwinLink(TM) (BPPD 2010). BPPD reviewed BCS' response and additional information and concludes that five of the eight deficiencies were satisfactorily addressed and resolved (U.S. EPA 09/28/10). In BCS' response to deficiency 8, BPPD recommends including the following:  "reporting resistance to Agency within 30 days" and "a long-term IRM action plan" as outlined below. The applicant stated that the remaining deficiencies (#2 and #3) would be addressed during the next cotton growing season. BPPD recommends that these two items be included as terms of registration.

A) Discussion regarding Deficiency 1:

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

BCS' response: In high-dose verification experiments (method 3 and 4), BCS reported that the mortality was calculated as follows: Mortality = 1- number of large larvae/ number of entry holes.

BPPD response: BPPD has verified the calculations and confirmed the reported mortality results. This deficiency has been satisfactorily addressed and resolved. BPPD concludes that the single Events Cry1Ab and Cry2Ae in cotton both express a less than high-dose against PBW and that TwinLink(TM) expresses a high-dose against PBW.

B) Discussion regarding Deficiencies 2-3:

Deficiency 2: In the modeling analysis provided by BCS, only cotton bollworm (CBW) was modeled but not Tobacco budworm (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.

BCS' response: BCS informed BPPD that an additional high-dose study for TBW is scheduled for the next cotton growing season when field infestations are present. Modeling resistance evolution in TBW to TwinLink(TM) cotton will be conducted afterwards incorporating dose profile and Natural Refuge assumptions.

BPPD response: BPPD will review the dose data and modeling for TBW in support of a Natural Refuge request when the new data become available. In the meantime, in order to pursue registration with a structured refuge for TwinLink(TM), as proposed by BCS during the June 21 teleconference, this additional information is not required.

C) Discussion regarding Deficiencies 4-6:

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). BPPD cannot verify the results of the artificial infestation experiments, however, 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 also 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(TM) Cotton. 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).

BCS' response: The most likely R-allele value for Cry1Ab was actually set at 0.002 in the original submission; however, the initial allele frequency was set at 0.00001. In order to address BPPD's concerns, BCS increased the initial R-allele frequency of Cry1Ab to 0.0001. With respect to incorporating Cry1Ab corn in the modeling landscape, all corn crops were converted to 80% Bt (dual gene) corn and 20% non-Bt corn. Mortality rates were based on H. zea data on TwinLink(TM). Results after 2000 simulations (with revised parameters) showed that resistance did not evolve to either toxin in any of the simulations. The rate of increase over the first five years for each locus reflected the presence of fitness costs. In many cases, particularly with the Cry1Ab resistance locus, the resistance allele frequency actually dropped. This was likely due to the recessive nature of the fitness costs simulated. The most likely initial resistance allele frequency was 10-fold greater for this locus than for the Cry2Ae locus resulting at least initially in greater expression of fitness costs. The recessive nature of fitness costs may explain why the final resistance allele frequencies at both loci were similar despite the fact that the most likely value for the initial Cry1Ab locus frequency was 10-fold greater. Informally, several runs were made without the Cry2Ae toxin to compare the rate of increase of the Cry1Ab resistance allele in a single gene versus a two gene construct, and the mean rate of increase was greater in the single gene case. These results support those of Gould et al. (2006) that not only do dual gene plants increase the expected longevity of toxins but they also increase the delay due to fitness costs.

Based on these results, there may be some criticism about the assumption of recessive inheritance of fitness costs given the lack of extensive genetic data. This assumption is, however, conservative. If fitness costs are additive or even dominant, those fitness costs would be expressed at even lower resistance allele frequencies. The current assumption allowed resistance allele frequencies to increase to higher levels before fitness costs were expressed. The fact that resistance did not evolve in these simulations despite the conservative assumption provides stronger evidence that the risk of resistance evolving over a 30 year time horizon is small.

BPPD response:  The additional modeling information has been reviewed and BPPD concludes that the deficiency has been adequately addressed and resolved. BCS was able to demonstrate that with more conservative (greater) resistance allele frequencies and the inclusion of Cry1Ab corn, TwinLink(TM) corn had a durability of more than 30 years.

D) Discussion regarding Deficiency 7:

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.

BSC response: For TBW and CBW, 10-20 population will be collected from the Cotton Belt; for PBW, 4-6 populations will be collected from the western USA (Arizona, New Mexico, West Texas, and California) during 2011. These samples in conjunction with data collected prior to commercialization will help establish the baseline susceptibility for all target pests. Resistance monitoring will focus on areas of highest adoption and highest risk for TBW and CBW and states where PBW are an economic pest. Where the risk is highest for resistance evolution in the target pests, BCS will collect at least 20 populations. Monitoring reports will include sampling methodology, bioassay methodology, standardized procedures, detection technique and sensitivity, statistical analysis of probability to detect resistance. Discriminating dose assays for TBW, CBW, and PBW will be provided to the Agency on a yearly basis.

BPPD response: BPPD concludes that BCS' monitoring plan reflects the paradigm established for other Bt cotton registrations and the deficiency has been satisfied. However BPPD recommends that in the Terms and Conditions of the registration, BCS be required to submit baseline susceptibility and a diagnostic assay data.

E) Discussion regarding Deficiency 8:

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.

BSC response: For PBW, BSC will use the remedial action plan developed by the Arizona Bt Cotton Working Group. For TBW and CBW, baseline susceptibility data from 2011 and 2012 will define aspects of "suspected" and "confirmed" resistance and field response for each pest. In general, the plan will incorporate the following measures to identify or confirm suspected resistance:

   * Monitoring (as outlined in MRID 47640014);
   * Education of customer with respect to monitoring of unexpected levels of damage, reporting suspected resistance, and applying established remedial insecticide treatments;
   * Confirm resistance when unexpected damage or survival exceed established upper limit of normal baseline levels, LC50/LC99 exceed established upper limit of baseline levels, and resistance is heritable;
   * Alternate methods to control pests as outlined under "Containment" below;
   * Sales restrictions/suspension until data provided and reviewed by EPA support that target pests have regained susceptibility to Bt proteins;
   * Containment through chemical insecticide spraying, accelerated harvest, crop destruction methods, crop rotation, tillage, use of sterile insects, use of products with different modes of action;
   * Monitoring will be increased in affected and surrounding areas; change in type and size of refuge should be considered.

BPPD response: BPPD has reviewed BCS's additional remedial action information and notes that the above is short summary of the applicant's actual response. BPPD noted that the remedial action plans needs to explicitly state that "the Agency is informed within 30 days of confirmed resistance". In addition, BCS should incorporate that "a long-term resistance management action plan will be designed according to characteristics of resistance events and local agronomic practices". If BCS makes these changes, then Deficiency 8 and the Agency's request for more information will have been satisfied.

Appendix D. III  -  Risk Assessment of data/information to respond to remaining IRM deficiencies.
Below is U.S. EPA's review (June 22, 2011) of BCS' resubmissions for FIFRA section 3 application for TwinLink(TM) Cotton expressing a pyramid with Events T304-40 and GHB 119 (MRIDs 48480011, 48480010, and 48495101), EPA File Symbols 264-RNOA and 264-RNOL: PC Codes: 006525 (Cry1Ab), 006600 (Cry 2Ae). In the original IRM risk assessment dated April 22, 2010, EPA identified eight deficiencies, five of which were addressed on 09/28/10. BCS provided additional data/information to satisfy the remaining deficiencies.

BPPD reviewed BCS' submission to address deficiencies noted in the original review of TwinLink(TM) Cotton, a two-gene pyramided PIP targeting Heliothine pests (BPPD 2010). BPPD's conclusions and recommendations are contained in this memo.

CONCLUSIONS AND RECOMMENDATIONS

DOSE VERIFICATION FOR TBW (Deficiency 3)

BCS provided enough data to resolve concerns with the dose for TwinLink(TM) Cotton and its single parental events against TBW, and Deficiency 3 has been addressed in a satisfactory manner. Specifically, based on the field experiment conducted with young cotton plants, BPPD concludes that Cry1Ab and Cry2Ae PIPs express a high dose against Heliothis virescens (TBW) during the early cotton growing season (also late season for Cry2Ae). No larvae on Cry2Ae and Cry1Ab cotton survived or developed to 3[rd] instar during the 11-day test duration, while 2.8% and 4.5% larvae in control treatments reached or surpassed the 3[rd] instar developmental stage. The lab experiment conducted with older cotton plants, however, resulted in 2.17% survival (> 3[rd] instar) on Cry1Ab plants expressing the parental event only suggesting that expression of Cry1Ab was decreased in older plants compared to younger plants. Based on the limited data available at this point, BPPD cautions on the side of error and concludes that Event T304-40 does not express a high-dose against TBW during the late cotton-growing season (based on larval mortality on older cotton plants in the lab). TwinLink(TM) cotton, however, expresses an effective high dose against TBW, and no larval survival was observed in the lab experiment.

MODELING FOR TBW AND TWINLINK(TM) COTTON WITH NATURAL REFUGE (Deficiency 2)

BCS provided additional modeling to resolve concerns for their "Natural Refuge" request for TwinLink(TM) Cotton, and Deficiency 2 has been addressed in a satisfactory manner. BPPD concludes that TwinLink(TM) Cotton should not pose a greater risk of resistance evolution in TBW than, for example, Bollgard II Cotton based on the modeling simulations provided in BCS submission.

Specifically, BCS conducted two modeling analyses (stochastic, spatially explicit and deterministic, spatially implicit) and generally took a conservative approach to modeling the durability of TwinLink(TM) targeting TBW. Mainly, the model landscape accounted for other Cry1A products and model explored several scenarios with increased dominance and lower survival based on the lower protein expression for Cry1Ab cotton based on lab observations. The natural refuge assumptions were based on available natural host information of Mississippi  -  a worst-case candidate state for this IRM strategy. The deterministic simulations estimated that durability of TwinLink(TM) Cotton would be as low as 80 years when all worst-case assumptions were incorporated or as high as 224 years using benchmark assumptions. This represents a 2.8 fold difference between estimated durabilities. The spatially explicit, stochastic simulations estimated that the durability for TwinLink(TM) Cotton would be on average between 20 years (possibly an unrealistic worst-case scenario based on survival percentages used) and 23 years (possibly a reasonable worst-case scenario). Assuming benchmark parameter values for mortality (as used for other Cry1A products in the past), the mean time to estimated product failure (resistance) was 26 years. Whether results differed significantly between any scenarios modeled was unclear since no such analysis and/or explanation accompanied the report. Additionally, comparative analysis between Bollgard II (Monsanto released data used for the natural refuge SAP) and TwinLink(TM) Cotton showed that estimated mean durabilities were comparable.

Although the likelihood for cross-resistance between Cry1Ab and Cry2Ae cotton has been shown to be small, BPPD notes that it would have been more conservative to consider and incorporate a small degree into the modeling analysis of TwinLink(TM) Cotton. Other forms of epistasis (expression of a gene is suppressed by a gene at another locus) were not considered: rather the applicant assumed that survival of genotypes was multiplicative for both loci (the least conservative assumption). The SAP (2011) suggested that other forms of epistasis should be explored for pyramided PIPs to address the risk of resistance evolution (see conclusion section for discussion).

Resistance evolved almost simultaneously to both toxins (under worst case assumptions) when lower protein expression for Cry1Ab was incorporated for older plants and raised some concerns with BPPD. Further analysis should be conducted to assess whether this outcome was an artifact of the modeling design taken by the modeler or a function of real temporal asynchrony from the growing oviposition period between generations and delayed oviposition of adults dispersing from senescing wild hosts to other acceptable hosts. BPPD notes though that the simulations with a lower dominance value for Cry1Ab did not result in simultaneous resistance evolution of the two toxins during the ten replications. It will be important to determine whether this outcome can be replicated for the durability of other pyramided cotton PIPs that express Cry1A Bt toxins which have lower expression levels during the later cotton growing season (resulting in greater dominance values for the target pest).

BPPD concludes that TwinLink(TM) Cotton deployed with a natural refuge strategy should not be expected to pose a greater risk of resistance evolution in TBW than for example Bollgard II Cotton based on the modeling simulations provided in BCS' submission.

   I. BACKGROUND

TwinLink(TM) cotton is a pyramided transgenic cotton trait that expresses Cry1Ab (Event T304-40), and Cry2Ae proteins (Event GHB119), and a glufosinate ammonium (herbicide) tolerant trait based on LibertyLink technology. TwinLink(TM) was developed by BCS, using conventional breeding techniques. The Cry1 and Cry2 Bt toxin group are effective against lepidoptera 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.

On April 22[nd], 2010, the BPPD IRM team completed its initial review of BCS' submission for a FIFRA product registration for TwinLink(TM) cotton. During its review, BPPD identified eight IRM deficiencies for which BCS needed to provide clarifications and/or data.

On July 29[th], 2010, BCS submitted their response to BPPD's eight listed IRM deficiencies for TwinLink(TM) (BPPD 2010a). BPPD reviewed BCS' response and additional information and concluded that five of the eight deficiencies were satisfactorily addressed and resolved (BPPD 2010b). The applicant stated that the remaining deficiencies (simulation modeling and second high-dose verification study, both for Tobacco budworm) would be addressed during the next cotton growing season. The third deficiency (#8) related to the remedial action plan and highlighted that BCS needed to explicitly state that "the Agency is informed within 30 days of confirmed resistance". This BPPD review assesses deficiencies 2 and 3 listed below:

      Deficiency 2: In the modeling analysis provided by BCS, only cotton bollworm (CBW) was modeled but not Tobacco budworm (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 either 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.

   II. BAYER CROPSCIENCE RESUBMISSION MATERIALS

                     1. Field Dose Study (MRID 48480010)

In 2011, a study was conducted in Mississippi cotton fields (MRID 48480010) to estimate mortality of Heliothis virescens (Tobacco budworm, TBW) on caged plants expressing parental Events Cry1Ab and Cry2Ae as well as the pyramided PIP.

Materials and Methods:

Four treatments were tested: 1) Cry1Ab cotton (T304-40), 2) Cry2Ae cotton (GHB119), 3) TwinLink(TM) cotton (T304-40 x GHB119), and 4) Coker 312 (negative control). A treatment plot consisted of two 38 inch rows of 40 feet in length (0.0029 acres) and each contained approximately 240 plants. Fine mesh netting cages were set up (8 ft x 20 ft) in each plot which covered approximately 120 plants. Artificial infestations were conducted with individuals from a laboratory strain from USDA-ARS, Stonesville (MS). Fifty males and 50 females were released into each cage on two dates separated by one month (two replicates). Mated females oviposited onto cotton plants, and first data collections began four days after introducing the adults into the cages to obtain lower limits of eggs deposited. Ten randomly selected plants in each cage were inspected and scored using binomial decision rule. If a plant contained at least 30 eggs, it was scored with a `yes'. Larval survival was estimated on 20 randomly selected plants within each cage, and survival and developmental stage were recorded. Larvae that survived 11 days of exposure on the plant and had developed to 3[rd] instar were scored as survivors. The lower limit of the proportion of surviving individuals was then computed by assuming that each plant was infested with 30 eggs (actual infestation was higher  -  data not shown here) yielding an initial egg infestation cohort of 600 eggs (20 plants x 30 eggs) per replicate of Bt plant type.

Results:
One larva developed to 1[st] instar on one Bt plant type (Cry1Ab), while 17 and 37 developed to >=3[rd] instar in the negative control plots (first and second replicate, respectively). Since no larvae survived on the three Bt treatments, there was no need to compute corrected mortality.

Table D.III.1. TBW larval survival after 11 days of exposure to TwinLink(TM) and its parental Events
Treatment Type
                  Number of Eggs (initial cohort  -  minimum)
                           Total Number of Survivors
                              Number of Survivors
                              (>= 3[rd] instar)
                   Proportion of Survivors  -  minimum value
                              (>= 3[rd] instar)
Coker 312 (Control)
    Replicate 1
                                      600
                                      31
                                      17
                                    0.0283
    Replicate 2
                                      600
                                      28
                                      27
                                    0.0450
Cry1Ab  (single Event)
    Replicate 1
                                      600
                                       1
                                       0
                                       0
    Replicate 2
                                      600
                                       0
                                       0
                                       0
Cry2Ae (single Event)
    Replicate 1
                                      600
                                       0
                                       0
                                       0
    Replicate 2
                                      600
                                       0
                                       0
                                       0
Cry1Ab x Cry2Ae (TL)
    Replicate 1
                                      600
                                       0
                                       0
                                       0
    Replicate 2
                                      600
                                       0
                                       0
                                       0
This Table has been modified from the Table in the original submission (p.12, MRID 48480010)

Applicant Conclusions:

"This study provided additional evidence for the high susceptibility of TBW to TwinLink(TM) as well as its parental (single) events. No survivors (>= 3[rd] instar) were found on the parental event, therefore, TwinLink(TM) should provide IRM benefits comparable to or exceeding those of other currently registered pyramided Bt cotton products approved for the natural refuge strategy."

                     2.       Greenhouse Dose Study (MRID 48480011)

In 2011, BCS conducted a greenhouse trial to provide further evidence that TwinLink(TM) Cotton and its parental events express high dose against Heliothis virescens.

Materials and Methods:

Cotton plants (144-184 days old) were placed into four isolated screened enclosures to hold groups of different Bt phenotypes. The treatments were 1) Cry1Ab cotton (T304-40), 2) Cry2Ae cotton (GHB119), 3) TwinLink(TM) cotton (T304-40 x GHB119), and 4) non-cotton plants (negative control). Each enclosure contained 12 plants of each treatment (three replicates total). Bt and non-Bt plants were artificially infested with 50 and 15 (24-hr old) TBW neonates, respectively. Larval counts were made at day 7 and 14 after infestation. Survival was computed as the proportion of individuals surviving 14 days and completing development to at least 3[rd] instar. Corrected mortality was calculated by dividing `proportional survival on Bt' by `proportional survival on non-Bt cotton'.

Results:

Larval survival on non-Bt and Cry1Ab plants was 69 and five 3[rd] instar larvae out of a total infestation with 540 (on control) and 1800 (on Bt); after correcting for control mortality, survival on Cry1Ab plants was estimated at 0.0217 (see Table 2). On Cry2Ae and TwinLink(TM) cotton plants, no survivors were found (infestation with 1800 larvae).

Table D.III. 5. TBW larval survival on parental Events and pyramided PIP TwinLink(TM) during the late cotton growing season
Treatment
                             Total Plants infested
                           Total Number of Neonates
                           Total Number of Survivors
                           Mean Proportion Survivors
                           Std. Deviation Proportion
                      Corrected Mean Proportion Survivors
Non-Bt
                                      36
                                      540
                                      69
                                     0.128
                                    0.0484

Cry1Ab
                                      36
                                     1800
                                       5
                                    0.00278
                                    0.00192
                                    0.0217
Cry2Ae
                                      36
                                     1800
                                       0
                                       0
                                       0
                                       0
TwinLink(TM)
                                      36
                                     1800
                                       0
                                       0
                                       0
                                       0
This Table has been modified from the Table in the original submission (p. 11, MRID 484800011

Conclusions:

The level of mortality observed in these trials and levels of survival on single-toxin plants was comparable to or exceeded that of the currently registered Bt cotton products. Lack of survivors on Cry2Ae plants underscores the highly effective dose of this protein in the TwinLink(TM) pyramid. The low level of survival has not been observed under field conditions but could be due to reduced protein expression levels in older plants tested (late-season decline has also been reported for Cry1Ac in currently registered Bt cotton products). This study shows that plants, even late in the production cycle, continue to cause very high mortality to TBW. This should provide additional support for the use of natural refuge strategy for T304-40 x GHB119.

                     3. Simulation Modeling for TwinLink(TM) Cotton and Tobacco Budworm (MRID 48495101)

BCS contracted the simulation modeling to Dr. Mike Caprio from Mississippi State University. Two different models, stochastic and deterministic, were employed to estimate the durability of TwinLink(TM) Cotton in an environment such as the state of Mississippi (worst-case state for natural refuge option).

Materials and Methods:

Spatially explicit, stochastic model:

This model was based on a previous model by Caprio et al. (2009) for Helicoverpa zea (cotton bollworm, CBW) and was modified to address TBW specifically and to conduct simulations for TwinLink(TM) cotton. Only the modifications to the model were described in BCS' modeling resubmission. A total of six different habitats were modeled (prevalence determined from 2002 data): Bollgard cotton, non-Bt cotton, wild hosts (clover, geranium), and TwinLink(TM) (post-cutout and very late season). Alternate hosts were modeled as separate patches, which in turn forced adult TBW to disperse between patches as wild hosts declined with progressing time. This landscape simulated a situation in the field where alternate hosts would be spatially segregated. All patches were randomly assigned the nature of plant type at the initiation of the simulation. All larvae and pupae were accounted for and placed as pupae in geranium patches at the beginning of each year. Because of complete mixing during the overwintering routine, the habitats were not redistributed at the beginning of each year. This allowed detecting the effects of habitat distribution between simulations.

The dose of Cry1Ab was modeled at 99.9% for the first three generations and subsequently reduced to address the greater survival seen on cotton plants in the lab expressing parental Cry1Ab. Four different levels of decline in protein expression were investigated leading to 0.1% survival (0% decline), 2% survival (level observed in lab), 5% (see Table 3 for genotypic survival), and 10% (extreme case). These levels of decline continued for two more generations until the overwintering subroutine began. Two values of dominance were used: 0.1 (high value) and 0.001 (lower value). Multiplicative toxin interactions were assumed. To overcome extinction of relatively small TBW populations, the fecundity was raised from 50 to 150 eggs/day-female in the model.

Table D.III.3. TBW Survival on TwinLink(TM) Cotton with 5% and 0.1% survival to Cry1Ab and Cry2Ae
Toxin
                                    Cry1Ab
                                    Cry1Ae
Genotypes
                                      SS
                                      RS
                                      RR

Pre-Cutout

SS
                                   0.000001
                                   0.0001009
                                     0.001

RS
                                   0.0001009
                                  0.01018081
                                    0.1009

RR
                                     0.001
                                    0.1009
                                      1.0

Post-Cutout
                                      SS
                                      RS
                                      RR

SS
                                    0.00005
                                   0.000145
                                     0.001

RS
                                    0.00504
                                    0.01463
                                    0.1009

RR
                                     0.05
                                     0.145
                                      1.0
Table modified from original Table (p.11, MRID 48495101)

Deterministic model:

The Caprio model (based on a construct previously employed by EPA-ORD) was used to model TBW resistance to TL cotton and modified to include refuges of variable size as well as Cry1Ab protein expression decline in older plants. Mortality from Cry2Ae was constant at 99.9% for each generation. For Cry1Ab, mortality was 99.9% for the first three generations (survival of the susceptible genotype was set at 0.0001) and declined thereafter to 98%. The natural refuge percentage varied for each generation and was 20%, 45%, 7%, 1%, 3%, and 3% for each consecutive generation (worst case simulations based on refuge data in worst case county of MS). Fitness for homozygous resistant individuals was 1.0 in all habitats (no fitness cost). Activity of the two toxins was multiplicative (no epistasis). Survivorship of the susceptible genotype was 0.0001% (0.01% x 0.01%). Dominance for TBW was set at 0.001 to compare modeling runs with other models for validation purposes. For further validation, the model was run for a single gene product (Cry1Ac such as for Bollgard), 20% refuge, and 50% reduction in adult emergence due to spraying. The estimated durability was 18.8 years. Given that Bollgard was used for almost 15 years, this would suggest that a dominance value of 0.001 may not be unreasonable and on the high side of realistic. Additionally, comparative modeling was conducted for Bollgard II (Monsanto released data for natural refuge SAP) and TwinLink(TM) to assess the relative durability of TwinLink(TM) to the already registered pyramided PIP.

Results:

Stochastic model: When dominance was set at 0.001, no resistance evolved to TwinLink(TM) within 31 years (including worst-case scenarios with 10% survival after cutout). When dominance was increased to 0.1, results were more complex (see Table 3). With 5% survival in the later season, mean time to resistance was 23.9 years. Greater survival later in the season resulted in faster resistance evolution. Additionally, in almost every simulation resistance to both toxins (Cry1A and Cry2Ae) occurred almost simultaneously. This was unexpected since Cry1Ab moved towards a lower dose later in the growing season, and the expectation was that resistance would evolve to Cry1Ab first. Analysis of data revealed that resistant individuals in two fields became temporarily isolated from the remaining refuge population early in the season. Toxin expression was high at that time and hence, only individuals resistant to both toxins survived. Their homozygous resistant offspring emerged before susceptible individuals dispersed into those fields. This resistance was not caused by isolation as described by Peck et al. (1999) but took place in two separate fields (far enough apart so no dispersal occurred between them) after complete mixing had occurred during the overwintering season (an assumption made by the modeler as described in the methodology section). This apparent temporal asynchrony occurred although no developmental delays were incorporated into the model and was a function of 1) a growing oviposition period between generations (adults emerge over an interval; late emerging cohorts will always be late until redistribution occurs during overwintering season) and 2) adults from senescing wild hosts had to disperse first to a new habitat before oviposition could take place (evidence for this phenomena from shorter "tail" of pupal emergence distribution from wild hosts compared to distributions from TL cotton). The data suggest that resistance evolved in "the tails of the generational distributions". Resistance observed under these model parameters was not specific to TwinLink(TM) Cotton but could be generated by a hypothetical two-gene PIP consisting of two high dose toxins (99.9% mortality). The modeler contended that the result might be an artifact based on model design aimed at addressing TBW biology. Further research, independent of any particular PIP, may be warranted.

The model was run using Monsanto's parameter values and dominance values of 0.001 and 0.1. With lower dominance, resistance did not evolve in any of the 10 replicates; with lower dominance values, resistance evolved as early as in 10.1 years and mean time to resistance was 27.3 years (comparable to TwinLink(TM) results).

Table D.III.6. Mean time to resistance for TBW to TwinLink(TM) Cotton
                        Survival to Cry1Ab Post-Cutout
                         Mean Time to Resistance (yrs)
                             Number of Simulations
                                    0.1%[1]
                                     26.0
                                      10
                                     2%[2]
                                     23.5
                                      10
                                      5%
                                     23.9
                                      10
                                      10%
                                     20.6
                                      10
Survival to Cry2Ae was held constant during the different simulations
[1] default survival value used by registrants based on field data from Cry1A plants prior to cutout.
[2] Survival value observed by BCS in 2011 greenhouse studies of plants after cutout.
Table modified from original Table (p.12, MRID 48495101)

Deterministic Model: The deterministic runs of the model resulted in an estimated 224 years before resistance evolved in TBW to TwinLink(TM) (both toxins) under benchmark assumptions. When the dominance value was increased to 0.01 (for later generations of TBW in response to lower expression levels in older cotton plants), it took 80 years before resistance evolved to TwinLink(TM) (see Table D.III.5 below). Unlike other previous models for TBW and currently registered Bt cotton PIPs expressing Cry1A protein, BCS incorporated a seasonal decline in Cry1Ab protein expression, which somewhat elevated the risk of resistance evolution.

Table D.III.5. Durability of TwinLink(TM) Cotton based on the deterministic model simulations
                          Dominance Value for Cry1Ab
                           Time to Resistance (yrs)
                                     0.001
                                      224
                                    0.01[1]
                                      80
TL simulations run with parameter values as close to Monsanto's values for Bollgard II
[1] Dominance is 10x that of Monsanto's value

Conclusions:

"Based on both stochastic and deterministic models, using worst-case scenarios for refuge size and contrasts of other key model parameters, TwinLink(TM) would not be expected to change the risk of resistance over existing dual-Bt products. Risk of resistance to TwinLink(TM) and all other Cry1A-based dual-Bt products registered in the US may be somewhat higher than indicated by previous simulations, owing to possible late-season decline in Cry1Ac expression."

   III. BPPD REVIEW AND CONCLUSIONS

DOSE (Deficiency 3): Based on the field experiment conducted with young cotton plants, BPPD concludes that Cry1Ab and Cry2Ae (individual parental events) express a high dose against Heliothis virescens (TBW) during the early cotton growing season (also late season for Cry2Ae). No larvae on Cry2Ae and Cry1Ab survived or developed to 3[rd] instar during the 11-day test duration, while 2.8% and 4.5% larvae in control treatments reached or surpassed the 3[rd] instar developmental stage. The lab experiment conducted with older cotton plants, however, resulted in 2.17% survival (> 3[rd] instar) on Cry1Ab plants expressing the parental event only suggesting that expression of Cry1Ab protein was decreased in older plants compared to younger plants. Based on the limited data available at this point, BPPD cautions on the side of error and concludes that Event T304-40 does not express a high-dose against TBW during the late cotton-growing season (based on larval mortality on older cotton plants in the lab). TwinLink(TM) cotton, however, expresses an effective high dose against TBW, and no larval survival was observed in the lab experiment.

MODELING (Deficiency 2): BCS conducted two modeling analyses (stochastic spatially explicit and deterministic, spatially implicit) and generally took a conservative approach to modeling the durability of TwinLink(TM) Cotton targeting TBW. Dr. Mike Caprio from Mississippi State University incorporated other Cry1A products into the landscape and explored several scenarios with increased dominance and lower survival based on the lower protein expression observed in the lab for Cry1Ab cotton. The natural refuge assumptions in the model were based on available host data of Mississippi - a worst-case candidate state for this IRM strategy. The deterministic simulations estimated that durability of TwinLink(TM) Cotton would be as low as 80 years when all worst-case assumptions were incorporated or as high as 224 years using benchmark assumptions. This was a 2.8 fold difference between estimated durabilities. The spatially explicit, stochastic simulations estimated that the durability for TwinLink(TM) Cotton was on average between 20 years (probably an unrealistic worst-case scenario based on survival percentages used) and 23 years (probably reasonable worst-case scenario). Assuming benchmark parameter values for mortality (as used for other Cry1A products in the past), the mean time to estimated product failure (resistance) was 26 years. Whether results differed significantly between any scenarios modeled was unclear since no such analysis and/or explanation accompanied the report. Additionally, comparative analysis between Bollgard II (Monsanto released data 	presented to SAP for Natural Refuge assessment) and TwinLink(TM) Cotton showed that estimated mean durabilities were comparable between the two pyramided PIPs.

Although the likelihood for cross-resistance between Cry1Ab and Cry2Ae cotton has been shown to be small, BPPD notes that it would have been more conservative to consider and incorporate a small degree of epistasis (expression of a gene is suppressed by a gene at another locus) into the modeling analysis of Cotton. Other forms of epistasis were not considered: rather the applicant assumed that survival of genotypes was multiplicative for both loci (the least conservative assumption). The SAP (2011) suggested that additional forms of epistasis should be explored for pyramided PIPs such as, for example, developmentally restricted expression of low levels of Cry-protease and where resistance at the a locus could be determined by the most rapidly evolving locus, which could drag other resistance loci along and, thereby, increase the rate of resistance evolution beyond what would be estimated by a purely multiplicative approach.

The discussion as to why resistance evolved almost simultaneously to both toxins (with assumption of increased dominance) raised some concerns with BPPD. Further analysis should be conducted to assess whether this outcome was an artifact of the modeling design taken by the modeler or a function of real temporal asynchrony from the growing oviposition period between generations and delayed oviposition of adults dispersing from senescing wild hosts to other acceptable hosts. BPPD notes though that the simulations with a lower dominance value for Cry1Ab did not result in simultaneous resistance evolution of the two toxins during the ten replications. It will be important to determine whether this outcome can be replicated for the durability of other pyramided cotton PIPs that express Cry1A Bt toxins with lower expression levels during the later cotton growing season (resulting in greater dominance values for the target pest).

BPPD concludes that TwinLink(TM) Cotton deployed with a natural refuge strategy should not be expected to pose a greater risk of resistance evolution in TBW than, for example, Bollgard II Cotton based on the modeling simulations provided in BCS submission.

APPENDIX E: References

Insect Resistance Management

1. BPPD, 2010a. EPA review of BCS' insect resistance management plan for a section(3) full commercial registration request for TwinLink(TM) Cotton (EPA File Symbol 264-RNOU, MRIDs 4760014 and 47634823). Memorandum from JC Martinez to S. Bacchus and D. Greenway on April 4.

2. BPPD, 2010b. EPA Review of Bayer's Response to IRM Deficiencies noted in BPPD's risk assessment for TwinLink(TM) dated 4/22/2010 (EPA File Symbol 264-RNOU, MRIDs 4760014 and 47634823). Memorandum from JC Martinez to S. Bacchus and D. Greenway on July 29.

3. Caprio MA, CD Parker, and JC Schneider. 2009. Future fitness of female insect pests in temporally stable and unstable habitats and its impact on habitat utility as refuges for insect resistance management. Journal of Insect Science, Vol. 9(44): 1-10

4. MA Caprio. 2011. A risk assessment of TwinLink(TM) Cotton and Tobacco Budworm (Heliothis virescens) in the Midsouth. Bayer CropScience unpublished report submitted to the Agency, MRID 48495101.

5. Crickmore  N, DR Zeigler, J Feitelson, E Schnepf, D Van Rie, D Lereclus, J Baum , and DH Dean. 1998. Revisions of the Nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, Vol. 62 (3): 807-813.

6. EPA Scientific Advisory Panel (SAP). 2011. Transmittal Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held December 8-9, 2010 to Address Scientific Issues Associated with Insect Resistance Management for SmartStax(TM) Refuge-in-the-Bag, a Plant-Incorporated Protectant (PIP) Corn Seed Blend. Memorandum from Dr. Sharlene Matten (DFO) to Dr. Steven Bradbury (Director of OPP) on March 3, 2011.

7. Navacerrad JJ, J Pierson, I Coats, and PL Hunst. 2011. High dose study: estimating the mortality of Heliothis virescens in a greenhouse on plants on T304-40 x GHB119 (TwinLink(TM)) Cotton (BCS-GH004-7 x BCS-GH005-8) and parental Event plants T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Bayer CropScience unpublished report submitted to the Agency, MRID 48480011

8. W Mullins. 2011. High dose study: estimating the mortality of Heliothis virescens in a Mississippi cotton field on caged plants on T304-40 x GHB119 (TwinLink(TM)) Cotton (BCS-GH004-7 x BCS-GH005-8) and parental Event plants T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Bayer Crop Science unpublished report submitted to the Agency, MRID 48480010

9. Peck SL, F Gould, and SP Ellner. 1999. Spread of resistance in spatially-extended regions of transgenic cotton: implications for management of Heliothis virescens (Lepidoptera: Noctuidea). Journal of Economic Entomology, Vol. 92: 1-1
REFERENCES

Other References:

U.S. EPA. 1998. FIFRA Scientific Advisory Panel Subpanel on Bacillus thuringiensis (Bt) Plant-Pesticides and Resistance Management, February 9 and 10, 1998

U.S. EPA. 2000. FIFRA Scientific Advisory Panel Subpanel on Bt Plant-Pesticides Risk and Benefit Assessments: Insect Resistance Management, October 18-20, 2000

U.S. EPA. 2001. Biopesticides Registration Action Document  -  Bacillus thuringiensis Plant Incorporated Protectants, http://www.epa.gov/oppbppd1/biopesticides/pips/bt_brad.htm

U.S. EPA. BPPD. 2009. Review of 2007 monitoring data submitted by ABSTC for lepidopteran Bt corn registrants (MRID 47413901). Memorandum from S. Borges to M. Mendelsohn dated April 8[th], 2009.

U.S. EPA REVIEWS  -  Insect Resistance Management

U.S. EPA. 2010a. Memorandum J. Martinez and A. Reynolds. BCS' Insect Resistance Management Plan for a Section (3) Full Commercial Registration Request of TwinLink Cotton (EPA Reg. No. 264-RNOU, MRIDs 4760014 and 47634823). April 22.

U.S. EPA. 2010b. Memorandum. J. Martinez and A. Reynolds. September 28, 2010. EPA Review of Bayer's Response to IRM Deficiencies noted in BPPD's risk assessment for TwinLink dated 4/22/2010 (EPA File Symbols 264-RNOU, 264-RNOL, and 264-RNOA, MRIDs 4760014 and 47634823). September 28.

U.S. EPA. 2011. Memorandum. J. Martinez and A. Reynolds. EPA review of Bayer CropScience's resubmissions for TwinLink(TM) Cotton sec(3) application expressing a pyramid with Events T304-40 and GHB 119 (MRIDs 48480011, 48480010, and 48495101), EPA File Symbol 264-RNOA, -RNOL: PC Codes: 006525 (Cry1Ab), 006600 (Cry 2Ae). June 28.

Appendix E.1. Bibliography of data submitted for registration of Bt Cry1Ab (Event T304-40) and TwinLink(TM) Cotton, its combination with Bt Cry2Ae (Event GHB119).
Most of the submissions below refer to Cry2Ae, and some of them also refer to TwinLink(TM). There may be repeats of those made for Cry2Ae in the Bibliography cited for Cry1Ab because they were submitted for TwinLink(TM), which contains both proteins.

I.	Cry 1Ab and TwinLink(TM) Cotton - Data Submissions
                                     MRID
                     Citation Reference  (Bt Cry 1Ab PIP)
47076900
Bayer Cropscience LP. (2007) Submission of Toxicity Data in Support of the Experimental Use of Bacillus thuringiensis Cry1Ab and Bacillus thuringiensis Cry2Ae for Use on Cotton. Transmittal of 2 Studies.
47076902
Rouque, D. (2006) Cry2Ae (GEM2) Protein: Acute Toxicity by Oral Gavage in Mice. Project Number: SA/06235, BBS06/010. Unpublished study prepared by Bayer Cropscience. 62 p.
47171700
Bayer Cropscience (2007) Submission of Residue Data in Support of the Experimental Use of Bacillus thuringiensis Cry1Ab Insecticidal Protein on Cotton Plants. Transmittal of 1 Study.
47171701
Hayes, R. (2007) Final Field Report for 2006 Season of Bacillus thuringiensis Cry1Ab Insecticidal Protein as Expressed in Cotton Plants. Project Number: 264/EUP/140. Unpublished study prepared by Bayer Cropscience. 6 p.
47634800
Bayer CropScience (2008) Submission of Efficacy Data in Support of the Application for Registration of BCS Cry1Ab Cotton Event T304-40. Transmittal of 20 Studies.
47634801
Ferullo, J. (2008) Product Characterization of Cry1Ab Cotton Event T304-40. Unpublished study prepared by Bayer BioScience N.V. 22 p.
47634802
Nennstiel, D. (2008) Product Characterization: Nutritional Characterization of Cry1Ab Cotton Event T304-40. Unpublished study prepared by Bayer CropScience LP. 8 p.
47634803
Currier, T.; Massengill, J. (2008) Protein Expression Analysis of Cotton Event T304-40, Expressing Cry1Ab and PAT/bar Proteins. Project Number: CP07B005. Unpublished study prepared by Bayer CropScience LP. 158 p.
47634804
Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of Cry1Ab Cotton Event T304-40 for PAT/bar and Cry1Ab and its Non-Transgenic Counterpart for PAT/bar and Cry1Ab Proteins. Project Number: CP07B011. Unpublished study prepared by Bayer CropScience LP. 154 p.
47634805
Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab and PAT/bar Proteins Produced in Escherichia coli to Cry1Ab and PAT/bar Proteins from Event T304-40 and TwinLink(TM) Cotton, Gossypium hirsutum. Project Number: CP08B003. Unpublished study prepared by Bayer CropScience LP. 52 p.
47634806
Nennstiel, D. (2008) ORF Analysis - Toxicology (Human Health Assessment). Unpublished study prepared by Bayer Cropscience. 10 p.
47634809
Stephenson, I. (2008) Cry1Ab Protein: Epitope Homology, N-glycosylation and Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens. Supplement to MRID 46708802 and MRID 46708803. Unpublished study prepared by Bayer BioScience N.V. 6 p.
47634812
Nennstiel, D. (2008) Toxicology (Human Health Assessment): History of Safe Use. Unpublished study prepared by Bayer BioScience N.V. 49 p.
47634813
Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of Risk of Gossypium hirsutum: Transformation Event T304-40 Expressing Cry1Ab Protein. Project Number: ECORA1. Unpublished study prepared by Bayer CropScience. 67 p.
47634815
Patnaude, M. (2008) Laboratory Study to Determine the Effects of Cry1Ab Protein on the Predatory Beetle Coleomegilla maculata. Project Number: EB99L008, 13798/6217. Unpublished study prepared by Springborn Smithers Laboratories. 60 p.
47634816
Scott, A. (2008) Cry1Ab Protein: Toxicity to Green Lacewing (Chrysoperla rufilabris). Project Number: EB99L009. Unpublished study prepared by Bayer CropScience LP. 4 p.
47634818
Bushey, D. (2008) Request for Waivers from the Requirement to Conduct a Non-Target Soil Invertebrate Study: Earthworm. Unpublished study prepared by Bayer CropScience LP. 6 p.
47634819
Sayers, L. (2008) Cry1Ab Protein: Ten Day Toxicity Test to Water Fleas (Daphnia magna) Under Static-Renewal Conditions. Project Number: 13798/6232, EB99L010. Unpublished study prepared by Springborn Smithers Laboratories. 45 p.
47634820
Martone, A. (2008) The Use of an Insect Heliothis virescens Bioassay to Determine the DT50 of the Cry1Ab Protein Produced from Escherichia coli After Aerobic Soil Degradation. Project Number: CP07B010. Unpublished study prepared by Bayer CropScience LP. 152 p.
47634821
Jesudason, P. (2008) Cry1Ab: Efficacy Assessment (Field and Laboratory). Unpublished study prepared by Bayer CropScience LP. 5 p.
47634822
Bushey, D. (2008) Request for a Waiver from the Requirement to Prepare a Public Interest Document. Unpublished study prepared by Bayer CropScience LP. 6 p.
47634823
Bushey, D. (2008) Request for a Waiver from the Requirement to Develop an Insect Resistance Management Plan. Unpublished study prepared by Bayer CropScience LP. 6 p.
47634824
Nennstiel, D. (2008) Detection Methods: rtPCR. Unpublished study prepared by Bayer BioScience N.V. 6 p.
47634825
Nennstiel, D. (2008) Protein Detection Methods: Lateral Flow Strip (LFS). Unpublished study prepared by EnviroLogix, Inc. 8 p.
47634826
Nennstiel, D. (2008) Protein Detection Methods: Enzyme Linked Immuno Sorbent Assay (ELISA). Unpublished study prepared by Strategic Diagnostics, Inc. and EnviroLogix, Inc. 18 p.
47640000
Bayer Cropscience LP (2008) Submission of Product Chemistry, Efficacy, Toxicity, Exposure and Risk and Public Interest Data in Support of the Application for Registration of BCS TwinLink(TM) Cotton Event T304-40 x GHB 119 Cotton. Transmittal of 14 Studies.
47640001
Ferullo J. (2008) TwinLink(TM) Product Characterization - Molecular Characterization. Unpublished study prepared by Bayer BioScience N.V. 6 p.
47640002
Jesudason, P. (2008) Product Characterization - Nutritional Characterization of Twinlink. Unpublished study prepared by Bayer Cropscience LP. 8 p.
47640003
Mackie, S. (2008) Protein Expression Analysis for T304-40, GHB119 and TwinLink(TM) Transgenic Cotton. Project Number: CP08B002, BBS07/013, BB06/010. Unpublished study prepared by Bayer Cropscience LP. 155 p.
47640004
Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of TwinLink(TM) Cotton for PAT/bar, Cry2Ae and Cry 1Ab and its Non-Transgenic Counterpart for PAT/bar, Cry2Ae and Cry1Ab Proteins. Project Number: CY07B008, BTS/0066/06, BBS07/006. Unpublished study prepared by Bayer Cropscience LP. 216 p.
47640005
Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and PAT/bar Proteins Produce in Escherichia coli and Bacillus thuringiensis to the Cry1Ab, Cry2Ae and PAT/bar Proteins from Events T304-40, GHB119 and TwinLink(TM) Cotton Seed, Gossypium hirsutum. USA, 2008. Project Number: CP08B011, BTS/0077/08, BBS07/006. Unpublished study prepared by Bayer Cropscience LP. 70 p.
47640006
Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry1Ab, Cry2Ae and PAT/bar in TwinLink(TM) Cotton, Gossypium hirsutum, USA, 2007. Project Number: CY07B006, BBS07/013, BIOX/001/PROTSUPP/07. Unpublished study prepared by Bayer Cropscience LP. 402 p.
47640007
Bushey, D. (2008) Toxicology: Summary Human Health Assessment. Unpublished study prepared by Bayer BioScience N.V. 8 p.
47640008
Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of Risk of TwinLink(TM) Cotton Expressing Cry1Ab and Cry2Ae Proteins. Project Number: ECORA3. Unpublished study prepared by Bayer Cropscience LP. 17 p.
47640009
Richards, K. (2008) Evaluation of the Dietary Effect(s) of Pollen from TwinLink(TM) on Honey Bee Larvae (Apis mellifera L.) Development. Project Number: EB99X037, CAR/204/08, BTS/0081/08. Unpublished study prepared by California Agricultural Research, Inc. and Bayer CropScience Ecotoxicology Department. 48 p.
47640010
Patnaude, M. (2009) Chronic Toxicity to Collembola (Folsomia candida) Using Transgenic Lyophilized TwinLink(TM) Cotton Leaf. Project Number: EB99X036, 13798/6223. Unpublished study prepared by Springborn Smithers Laboratories. 40 p.
47640011
Stafford, J. (2008) Broiler Chicken Nutritional Equivalency Study with TwinLink(TM) Cotton. Project Number: 13798/4118, TX99X106, M/310302/01/1. Unpublished study prepared by Springborn Smithers Laboratories. 223 p.
47640012
Rinehardt, M.; Holloway, J. ; Bushey, D. (2008) Agronomic Performance and Efficacy of TwinLink(TM) Cotton Obtained by Conventional Breeding of Cry1Ab Cotton Event T304-40 and Cry2Ae Cotton Event GHB119: 2007-2008 USA Production Seasons. Project Number: 07/044/102N, IR/7D/GH/MR, 07/059104N. Unpublished study prepared by Bayer Cropscience LP. 68 p.
47640013
Maclntosh, S.; Bushey, D.; Christian, M.; et al. (2008) Public Interest Document for Bacillus thuringiensis Cry1Ab and Cry2Ae Insecticidal Proteins as Expressed in Combined Trait Cotton (TwinLink(TM) Cotton) Plants. Unpublished study prepared by Bayer CropScience LP. 36 p.
47640014
Maclntosh, S.; Holloway, J.; Jansens, S.; et al. (2008) Insect Resistance Management Plan: TwinLink(TM) Cotton. Unpublished study prepared by Bayer CropScience LP. 88 p.
47651100
Bayer Cropscience LP - Bioscience (2008) Submission of Fate and Toxicity Data in Support of the Application for Registration of BCS Cry1Ab Cotton Event T304-40. Transmittal of 6 Studies.
47651101
Rouquie, D. (2007) Cry1Ab Protein: In Vitro Digestibility Study in Simulated Intestinal Fluid. Project Number: M/295264/01/1, SA/07111, LYNX/PSI/N/TX99L035. Unpublished study prepared by Bayer Cropscience. 54 p.
47651102
Rouquie, D. (2007) Cry1Ab Protein: In Vitro Digestibility Study in Human Simulated Gastric Fluid. Project Number: M/295272/01/1, SA/07110, LYNX/PSIN/TX51X006. Unpublished study prepared by Bayer Cropscience. 56 p.
47651103
Rouquie, D. (2007) Cry1Ab Protein: Acute Toxicity by Oral Gavage in Mice. Project Number: M/295507/01/1, SA/07109, LYNX/PSI/NTX51X007. Unpublished study prepared by Bayer Cropscience. 58 p.
47651104
Rouquie, D. (2007) Cry1Ab Protein: Heat Stability Study. Project Number: M/295690/01/1, SA/07112, LYNX/PSI/N/TX99L034. Unpublished study prepared by Bayer Cropscience. 51 p.
47651105
Richards, K. (2008) Evaluation of the Dietary Effect(s) of a Cry1Ab Protein on Honey Bee Larvae (Apis mellifera L.). Project Number: EB99X030, CAR/177/07, BBS07/006. Unpublished study prepared by California Agricultural Research, Inc. 111 p.
47651106
Patnaude, M. (2008) Chronic Toxicity to Collembola (Folsomia candida) Using Cry1Ab Protein. Project Number: 13798/6218, EB99X028, BBS07/006. Unpublished study prepared by Springborn Smithers Laboratories. 61 p.
47692700
Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS Cry1Ab Cotton Event T304-40. Transmittal of 1 Study.
47692701
Patnaude, M. (2009) Cry1Ab Protein - Toxicity to Green Lacewing (Chrysoperla rufilabris) Following OPPTS Guideline 885.4340. Project Number: 13798/6230, EB99L009, 082808. Unpublished study prepared by Springborn Smithers Laboratories. 48 p.
47850300
Bayer CropScience, LP (2009) Submission of Environmental Fate Data in Support of the Experimental Use of TwinLink(TM) Cotton on Cotton Plants. Transmittal of 1 Study.
47850301
Coats, I. (2009) TwinLink(TM) Cotton (BCS-GH004-7 x BCS-GH005-8): Investigation of Pollen Flow in Puerto Rico. Unpublished study prepared by Bayer CropScience, LP. 24 p.
47871400
Bayer CropSciences (2009) Submission of Product Chemistry, Environmental Fate and Toxicity Data in Support of the Application for Registration of BCS Cry1Ab Cotton Event T304-40. Transmittal of 4 Studies.
47871401
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Product Analysis Studies as Outlined in 40 CFR 158.2120-Cry1Ab. Unpublished study prepared by Bayer CropScience LP. 7 p.
47871402
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Toxicology Studies to Test for Health Effects as Outlined in 40 CFR 158.2140-Cry1Ab. Unpublished study prepared by Bayer CropScience LP. 9 p.
47871403
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Nontarget and Environmental Fate Studies to Test for Ecological Effects as Outlined in 40CFR 158.21-Cry1Ab. Unpublished study prepared by Bayer CropScience LP. 7 p.
47871404
Hunst, P. (2009) Bridging Data from TwinLink(TM) Cotton Avian Nutritional Study to Parental Events T304-40 and GHB119. Unpublished study prepared by Bayer CropScience LP. 7 p.
47871600
Bayer CropScience, LP (2009) Submission of Product Chemistry and Toxicity Data in Support of the Application for Registration of BCS TwinLink(TM) Cotton Event T304-40xGHB 119 Cotton. Transmittal of 6 Studies.
47871601
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Product Analysis Studies. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47871602
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Toxicology Studies to Test for Health Effects as Outlined in TwinLink(TM). Unpublished study prepared by Bayer CropScience. 9 p.
47871603
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Nontarget and Environmental Fate Studies to Test for Ecological Effects - TwinLink(TM). Unpublished studies prepared by Bayer CropScience. 7 p.
47871604
Currier, T. (2009) Protein Expression Analysis for Pat/bar, Cry1Ab and Cry2Ae Proteins in Whole TwinLink(TM) Plants Grown in the Field. Project Number: CP09Q005. Unpublished study prepared by Bayer CropScience, LP. 28 p.
47871605
Hunst, P. (2009) Investigation of Synergistic Effects of the Cry1Ab and Cry2Ae Proteins expressed in TwinLink(TM) Cotton (BCS-GH004-7 x BCS-GH005-8) on Target Insect Pests Using Leaf Bioassay. Unpublished study prepared by Bayer CropScience, LP. 8 p.
47871606
Coats, I. (2009) Supplement to MRID # 47640014: Insect Resistance Management Plan: TwinLink(TM) Cotton. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47903100
Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS TwinLink(TM) Cotton Event T304-40 x GHB 119 Cotton. Transmittal of 1 Study.
47903101
Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian Oral Toxicity Test: TwinLink(TM). Unpublished study prepared by Bayer CropScience LP. 16 p.
47903200
Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS CRY1AB Cotton Event T304-40. Transmittal of 1 Study.
47903201
Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian Oral Toxicity Test: TwinLink. Unpublished study prepared by Bayer CropScience LP. 16 p.
47928800
Bayer CropScience, LP-BioScience. (2009) Submission of Efficacy Data in Support of the Registration of BSC TwinLink(TM) Cotton Event T304-40 X GHB 119 Cotton. Transmittal of 1 Study.
47928801
Coats, I. (2009) Addendum to Product Chemistry for TwinLink: Structural and Functional Equivalence of Cry1Ab and Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry1Ab, Cry2Aa and PAT/Bar in TwinLink(TM) Cotton, Gossypium hirsutum, USA, 2007: Final Report. Project Number: CY07B006. Unpublished study prepared by Bayer CropScience, LP. 7 p.
48166300
Bayer CropScience LP-BioScience (2010) Submission of Efficacy Data in Support of the Experimental Use of Bacillus thuringiensis Cry2Ae Insecticidal Protein and Bacillus thuringiensis Cry1Ab and Cry2Ae Insecticidal Proteins on Cottons. Transmittal of 1 Study.
48166301
Bushey, D.; Acker, T.; Price, K.; et al. (2010) TwinLink(TM) Cotton (BCS-GH004-7 x BCS-GH005-8): Investigation of Pollen Flow in Puerto Rico: Year 2: 2009-2010. Project Number: M/386577/01/1. Unpublished study prepared by Bayer CropScience. 21 p.
48427900
Bayer CropScience (2011) Submission of Efficacy Data in Support of the Experimental Use of T304-40 x GHB119 x COT102 x COT67B in Cotton. Transmittal of 1 Study.
48427901
Robinson, T. (2011) Cry1Ab, Cry2Ae, PAT/bar, FLCry1Ab, Vip3Aa, APH4 Levels in Selected Tissues of the Combined Trait Product T304-40 x GHB119 x COT102 x COT67B (BCS-GHOO4-7 x BCS- GH005-8 x SYN-IR1O2-7 x SYN-IRO67-1). Project Number: 10/BM45L001. Unpublished study prepared by Bayer CropScience, LP. 22 p.
48471900
Bayer CropScience (2011) Submission of Product Chemistry Data in Support of the Experimental Use of Cry2Ae (GEM2) in Cotton. Transmittal of 1 Study.
48471901
Currier, T. (2011) Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton Leaves is Glycosylated: Final Report. Project Number: BK05B003. Unpublished study prepared by Bayer CropScience. 32 p.
48480000
Bayer CropScience LP (2011) Submission of Product Chemistry and Efficacy Data in Support of the Applications for Registration of BCS Cry1Ab Cotton Event T304-40, BCS Cry2Ae Cotton Event GHB119 and BCS TwinLink(TM) Cotton Event T304-40 x GHB 119 Cotton, and the Petition for Tolerance of Cry2Ae. Transmittal of 11 Studies.
48480001
Habex, V.; Verhaeghe, S. (2008) Detailed Insert Characterization of Gossypium hirsutum Transformation Event GHB119 by Southern Blot Analysis. Project Number: BBS07/009, M/308404/02/1. Unpublished study prepared by Bayer BioScience N.V. 43 p.
48480002
Moens, S. (2008) Confirmation of the Absence of Vector Backbone Sequences in Gossypium hirsutum Transformation Event GHB119. Project Number: BBS06/007, M/311127/02/1. Unpublished study prepared by Bayer BioScience N.V. 40 p.
48480003
Moens, S.; Criel, I. (2008) Detailed Insert Characterization of Gossypium hirsutum Transformation Event T304-40. Project Number: BBS07/014, M/311245/03/1. Unpublished study prepared by Bayer BioScience N.V. 62 p.
48480004
Habex, V.; Van Herck, H. (2007) Confirmation of the Absence/Presence of Vector Backbone Sequences in Gossypium hirsutum Transformation Event T304-40: Final Report. Project Number: BBS05/003, M/308400/02/1. Unpublished study prepared by Bayer BioScience N.V. 41 p.
48480005
Currier, T. (2011) Characterization of Cry1Ab and (Inert Ingredient) Proteins Produced in Event T304-40 Cotton, Gossypium hirsutum USA, 2011. Project Number: 10/BM99L508, M/406631/01/1. Unpublished study prepared by Bayer CropScience LP. 31 p.
48480006
Moens, S. (2011) Characterization of Cry2Ae and (Inert Ingredient) Proteins Expressed in Gossypium hirsutum Transformation Event GHB119. Project Number: BIO2/005/PROTEQUL/512, M/406615/02/1. Unpublished study prepared by Bayer BioScience N.V. 35 p.
48480007
Currier, T. (2011) Structural Stability Analysis of Gossypium hirsutum Combined Event TwinLink(TM) X GlyTol (GHB614): Final Report. Project Number: CP08Q012/2. Unpublished study prepared by Bayer CropScience LP. 34 p.
48480008
Currier, T.; Harbin, A. (2011) Protein Expression Analysis for GlyTol x TwinLink(TM) (GHB614 x T304-40 x GHB119) Cotton Grown in the Field, USA 2010. Project Number: 10BM99L440B, M/406245/01/1. Unpublished study prepared by Bayer CropScience LP. 125 p.
48480009
Bushey, D.; Jansens, S.; Becker, A. (2010) Insect Bioassay of Plant Material Expressing Cotton Events T304-40, GHB119, and TwinLink(TM) (T304-40xGHB119). Project Number: BIO2/005, INSECT/BIOASSAY/FROM/MONHEIM. Unpublished study prepared by Bayer CropScience. 22 p.
48480010
Mullins, W. (2011) High Dose Study: Estimating the Mortality of Heliothis virescens in a Mississippi Cotton Field on Caged Plants of T304-40 x GHB119 (TwinLink(TM)) Cotton (BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Events T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Unpublished study prepared by Bayer CropScience LP. 16 p.
48480011
Dennehy, T. (2011) High Dose Study: Estimating the Mortality of Heliothis virescens in a Greenhouse on Plants of T304-40 x GHB119 (TwinLink(TM)) Cotton (BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Event Plants T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Project Number: BA44Y001. Unpublished study prepared by Bayer CropScience LP. 16 p.
48495100
Bayer CropScience (2011) Submission of Efficacy Data in Support of the Application for Registration of the TwinLink(TM). Transmittal of 1 Study.
48495101
Dennehy, T. (2011) A Risk Assessment of TwinLink(TM) Cotton and Tobacco Budworm (Heliothis virescens) in the Midsouth. Unpublished study prepared by Mississippi State University. 15 p.

Appendix E.2. Bibliography of data submitted for Bacillus thuringiensis Bt Cry 2Ae (Event GHB119) and TwinLink (TM), its combination with Bt Cry1Ab (Event T304-40)

Most of the submissions below refer to Cry2Ae, and some of them also refer to TwinLink(TM) Cotton. There may be repeats of those made for Cry2Ae in the Bibliography cited for Cry1Ab because they were submitted for TwinLink(TM) Cotton, which contains both proteins.

                                     MRID
                      Citation Reference (Bt Cry 2Ae PIP)
46708900
Bayer Cropscience LP (2005) Submission of Product Chemistry, Toxicity and Efficacy Data in Support of the Experimental Use of Cry2Ae for Use on Cotton. Transmittal of 9 Studies.
46708901
Jansens, S.; Rouan, D. (2005) Characteristics of Cry2Ae Cotton Plants Derived From Transformation Events GHB119 and GHB714. Preliminary Report. Unpublished study prepared by Aventis Cropscience N.V. 24 p.
46708902
Habex, V. (2005) Description of the Amino Acid Sequence of the Cry2Ae Protein. Project Number: GEM2/AAS/01. Unpublished study prepared by Aventis Cropscience N.V. 5 p.
46708903
Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens. Project Number: SA/05133, TX99S005, B004973. Unpublished study prepared by Bayer Cropscience. 120 p.
46708904
Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Epitome Homology and N-Glycosylation Searches. Project Number: SA/05134, TX99S006, B004974. Unpublished study prepared by Bayer Cropscience. 192 p.
46708905
Rouquie, D. (2005) Cry2Ae (GEM2) Protein: In Vitro Digestibility Study in Simulated Gastric Fluid. Project Number: SA/05032. Unpublished study prepared by Bayer Cropscience. 44 p.
46708906
Rouquie, D. (2005) Cry2Ae (GEM2) Protein: Acute Toxicity by Intravenous Injection in the Mouse. Project Number: SA/05033, B004972. Unpublished study prepared by Bayer Cropscience. 80 p.
46708907
Currier, T. (2005) Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton Leaves in Glycosylated. Project Number: BK05B003, B004964. Unpublished study prepared by Bayer Cropscience LP. 28 p.
46708908
Saey, B.; Jansens, S. (2005) PAT Cry2Ae Protein in Cotton Tissues of Events GHB119 and GHB714: Preliminary Report. Unpublished study prepared by Aventis Cropscience N.V. 9 p.
46708909
Williams, D.; Jansens, S. (2005) Preliminary Efficacy Report of Cry2Ae Cotton Plants Derived from Transformation Events GHB119 and GHB714. Unpublished study prepared by Aventis Cropscience N.V. 6 p.
47076900
Bayer Cropscience LP. (2007) Submission of Toxicity Data in Support of the Experimental Use of Bacillus thuringiensis Cry1Ab and Bacillus thuringiensis Cry2Ae for Use on Cotton. Transmittal of 2 Studies.
47076902
Rouque, D. (2006) Cry2Ae (GEM2) Protein: Acute Toxicity by Oral Gavage in Mice. Project Number: SA/06235, BBS06/010. Unpublished study prepared by Bayer Cropscience. 62 p.
47125100
Bayer CropScience LP. (2007) Submission of Product Chemistry and Fate Data in Support of the Experimental Use of Bacillus thuringiensis Cry2Ae Insecticide Protein in Cotton. Transmittal of 2 Studies.
47125101
Jansens, S.; Rouan, D.; Habex, V. (2006) Characteristics of Cry2Ae Cotton Plants Derived from Transformation Events GHB119 and GHB714: Preliminary Report and Supplemental Information. Unpublished study prepared by Aventis Cropscience N.V. 29 p.
47125102
Rouquie, D. (2007) Cry2Ae (GEM2) Protein: In Vitro Digestibility Study in Simulated Gastric Fluid. Project Number: SA/07101, TX99L005, M/287164/01/1. Unpublished study prepared by Bayer Cropscience. 42 p.
47640000
Bayer Cropscience LP (2008) Submission of Product Chemistry, Efficacy, Toxicity, Exposure and Risk and Public Interest Data in Support of the Application for Registration of BCS TwinLink(TM) Cotton Event T304-40 x GHB 119 Cotton. Transmittal of 14 Studies.
47640001
Ferullo J. (2008) TwinLink(TM) Product Characterization - Molecular Characterization. Unpublished study prepared by Bayer BioScience N.V. 6 p.
47640002
Jesudason, P. (2008) Product Characterization - Nutritional Characterization of Twinlink. Unpublished study prepared by Bayer Cropscience LP. 8 p.
47640003
Mackie, S. (2008) Protein Expression Analysis for T304-40, GHB119 and TwinLink(TM) Transgenic Cotton. Project Number: CP08B002, BBS07/013, BB06/010. Unpublished study prepared by Bayer Cropscience LP. 155 p.
47640004
Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of TwinLink(TM) Cotton for PAT/bar, Cry2Ae and Cry 1Ab and its Non-Transgenic Counterpart for PAT/bar, Cry2Ae and Cry1Ab Proteins. Project Number: CY07B008, BTS/0066/06, BBS07/006. Unpublished study prepared by Bayer Cropscience LP. 216 p.
47640005
Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and PAT/bar Proteins Produce in Escherichia coli and Bacillus thuringiensis to the Cry1Ab, Cry2Ae and PAT/bar Proteins from Events T304-40, GHB119 and TwinLink(TM) Cotton Seed, Gossypium hirsutum. USA, 2008. Project Number: CP08B011, BTS/0077/08, BBS07/006. Unpublished study prepared by Bayer Cropscience LP. 70 p.
47640006
Martone, A. (2008) Structural and Functional Equivalence of Cry1Ab, Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry1Ab, Cry2Ae and PAT/bar in TwinLink(TM) Cotton, Gossypium hirsutum, USA, 2007. Project Number: CY07B006, BBS07/013, BIOX/001/PROTSUPP/07. Unpublished study prepared by Bayer Cropscience LP. 402 p.
47640007
Bushey, D. (2008) Toxicology: Summary Human Health Assessment. Unpublished study prepared by Bayer BioScience N.V. 8 p.
47640008
Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of Risk of TwinLink(TM) Cotton Expressing Cry1Ab and Cry2Ae Proteins. Project Number: ECORA3. Unpublished study prepared by Bayer Cropscience LP. 17 p.
47640009
Richards, K. (2008) Evaluation of the Dietary Effect(s) of Pollen from TwinLink(TM) on Honey Bee Larvae (Apis mellifera L.) Development. Project Number: EB99X037, CAR/204/08, BTS/0081/08. Unpublished study prepared by California Agricultural Research, Inc. and Bayer CropScience Ecotoxicology Department. 48 p.
47640010
Patnaude, M. (2009) Chronic Toxicity to Collembola (Folsomia candida) Using Transgenic Lyophilized TwinLink(TM) Cotton Leaf. Project Number: EB99X036, 13798/6223. Unpublished study prepared by Springborn Smithers Laboratories. 40 p.
47640011
Stafford, J. (2008) Broiler Chicken Nutritional Equivalency Study with TwinLink(TM) Cotton. Project Number: 13798/4118, TX99X106, M/310302/01/1. Unpublished study prepared by Springborn Smithers Laboratories. 223 p.
47640012
Rinehardt, M.; Holloway, J.; Bushey, D. (2008) Agronomic Performance and Efficacy of TwinLink(TM) Cotton Obtained by Conventional Breeding of Cry1Ab Cotton Event T304-40 and Cry2Ae Cotton Event GHB119: 2007-2008 USA Production Seasons. Project Number: 07/044/102N, IR/7D/GH/MR, 07/059104N. Unpublished study prepared by Bayer Cropscience LP. 68 p.
47640013
Maclntosh, S.; Bushey, D.; Christian, M.; et al. (2008) Public Interest Document for Bacillus thuringiensis Cry1Ab and Cry2Ae Insecticidal Proteins as Expressed in Combined Trait Cotton (TwinLink(TM) Cotton) Plants. Unpublished study prepared by Bayer CropScience LP. 36 p.
47640014
Maclntosh, S.; Holloway, J.; Jansens, S.; et al. (2008) Insect Resistance Management Plan: TwinLink(TM) Cotton. Unpublished study prepared by Bayer CropScience LP. 88 p.
47641900
Bayer CropScience (2009) Submission of Product Chemistry and Toxicity Data in Support of the Application for Registration of BCS Cry2Ae Cotton Event GHB119 Cotton and the Petition for Tolerance of Bacillus thuringiensis Cry2Ae. Transmittal of 26 Studies.
47641901
Ferullo, J. (2008) Product Characterization of Cry2Ae Cotton Event GHB119. Unpublished study prepared by Bayer BioScience N.V. 24 p.
47641902
Jesudason, P. (2008) Product Characterization - Nutritional Characterization of Cry2Ae Protein. Unpublished study prepared by Bayer CropScience. 6 p.
47641903
Currier, T. (2008) Protein Expression Analysis of Cotton Event GHB119, Expressing Cry2Ae and PAT/bar Proteins. Project Number: CY07B005. Unpublished study prepared by Bayer CropScience LP. 161 p.
47641904
Martone, A. (2008) Analyses of Raw Agricultural Commodity (Fuzzy Seed) of Cry2Ae Cotton Event GHB119 for PAT/bar and Cry2Ae and its Non-Transgenic Counterpart for PAT/bar and Cry2Ae Proteins. Project Number: CY07B001. Unpublished study prepared by Bayer CropScience. 123 p.
47641905
Martone, A. (2008) Structural and Functional Equivalence of Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry2Ae and PAT/bar Proteins in GHB119 Cotton, Gossypium hirsutum. Project Number CY07B002. Unpublished study prepared by Bayer CropScience. 57 p.
47641906
Capt, A.; Nennstiel, D. (2008) Toxicology (Human Health Assessment): Summary of Analysis of Open Reading Frame Homology Searches. Unpublished study prepared by Bayer Cropscience. 10 p.
47641907
Rouquie, D. (2008) Cry2Ae Protein: In Vitro Digestibility Study in Simulated Intestinal Fluid. Project Number: SA08127. Unpublished study prepared by Bayer Cropscience. 52 p.
47641908
Rouquie, D. (2008) Cry2Ae Protein with Transit Peptide Epitope Homology and N-Glycosylation Searches. Project Number: SA08209, TX99L003. Unpublished study prepared by Bayer Cropscience. 30 p.
47641909
Rouquie, D. (2008) Cry2Ae Protein with Transit Peptide Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens. Project Number: SA08208, TX99L004. Unpublished study prepared by Bayer Cropscience. 444 p.
47641910
Stephenson, I. (2008) Update on Cry2Ae Protein: In Vitro Digestibility Study in Human Simulated Gastric Fluid, Overall Amino Acid Sequence Homology Search with Known Toxins and Allergens, and Epitope Homology and N-Glycosylation Searches. Unpublished study prepared by Bayer BioScience N.V. 6 p.
47641911
Rouquie, D. (2008) Cry2Ae Protein: Heat Stability Study. Project Number: SA08128, TX99L036. Unpublished study prepared by Bayer Cropscience. 49 p.
47641912
Nennstiel, D. (2008) Cry2Ae Protein: History of Safe Use. Unpublished study prepared by Bayer BioScience N.V. 7 p.
47641913
Chalmers, A. (2008) Summary of Non-Target Organism Testing and Assessment of Risk of Gossypium hirsutum: Transformation Even GHB119 Expressing Cry2Ae Protein. Project Number: ECORA2. Unpublished study prepared by Bayer CropScience LP. 50 p.
47641914
Richards, K. (2008) Evaluation of the Dietary Effect(s) of a Cry2Ae Protein on Honey Bee Larvae. Project Number: EB99L001, CAR178/07. Unpublished study prepared by California Agricultural Research, Inc. 109 p.
47641915
Patnaude, M. (2007) Laboratory Study to Determine the Effects of Cry2Ae Protein on the Predatory Beetle Coleomegilla maculata. Project Number: EB99X006, 13798/6203. Unpublished study prepared by Springborn Smithers Laboratories. 64 p.
47641916
Scott, A. (2008) Cry2Ae Protein - Toxicity to Green Lacewing (Chrysoperla rufilabris). Unpublished study prepared by Bayer CropScience LP. 4 p.
47641917
Patnaude, M. (2008) Chronic Toxicity to Collembola (Folsomia candida) using GEM2 Proteins. Project Number: EB99X0081, 13798/6204. Unpublished study prepared by Springborn Smithers Laboratories. 133 p.
47641918
Patnaude, M. (2008) Cry2Ae Protein - Acute Toxicity to Earthworms (Eisenia fetida). Project Number: 13798/6222, EB99X032. Unpublished study prepared by Springborn Smithers Laboratories. 37 p.
47641919
Sayers, L. (2008) Cry2Ae Protein - Ten Day Toxicity Test to Water Fleas (Daphnia magna) Under Static-Renewal Conditions. Project Number: 13798/6233, EB99X033. Unpublished study prepared by Springborn Smithers Laboratories. 45 p.
47641920
Martone, A. (2008) The Use of an Insect Heliothis virescens Bioassay to Determine the DT50 of the Cry2Ae Protein Produced from Bacillus thuringiensis after Aerobic Soil Degradation. Project Number: CY07B007. Unpublished study prepared by Bayer CropScience LP. 152 p.
47641921
Jesudason, P. (2008) Cry2Ae: Efficacy Assessment (Field and Laboratory). Unpublished study prepared by Bayer CropScience LP. 5 p.
47641922
Bushey, D. (2008) Request for a Waiver from the Requirement to Prepare a Public Interest Document (Cry2Ae). Unpublished study prepared by Bayer CropScience LP. 6 p.
47641923
Bushey, D. (2008) Request for a Waiver from the Requirement to Develop an Insect Resistance Management Plan (Cry2Ae). Unpublished study prepared by Bayer CropScience LP. 6 p.
47641924
Nennstiel, D. (2008) Detection Methods: rtPCR (Cry2Ae). Unpublished study prepared by Bayer BioScience N.V. 7 p.
47641925
Nennstiel, D. (2008) Protein Detection Methods: Lateral Flow Strip (Cry2Ae). Unpublished study prepared by EnviroLogix Inc. 8 p.
47641926
Nennstiel, D. (2008) Protein Detection Methods: Enzyme Linked Immuno Sorbent Assay (ELISA) (Cry2Ae). Unpublished study prepared by EnviroLogix, Inc. 10 p.
47692500
Bayer CropScience (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS Cry2Ae Cotton Event GHB119 Cotton. Transmittal of 1 Study.
47692501
Patnaude, M. (2009) Cry2Ae Protein - Toxicity to Green Lacewing (Chrysoperla rufilabris) Following OPPTS Guideline 885.4340. Project Number: 13798/6229, EB99X025, BBS07/007. Unpublished study prepared by Springborn Smithers Laboratories. 65 p.
47871400
Bayer CropSciences (2009) Submission of Product Chemistry, Environmental Fate and Toxicity Data in Support of the Application for Registration of BCS Cry1Ab Cotton Event T304-40. Transmittal of 4 Studies.
47871403
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Nontarget and Environmental Fate Studies to Test for Ecological Effects as Outlined in 40CFR 158.21-Cry1Ab. Unpublished study prepared by Bayer CropScience LP. 7 p.
47871404
Hunst, P. (2009) Bridging Data from TwinLink(TM) Cotton Avian Nutritional Study to Parental Events T304-40 and GHB119. Unpublished study prepared by Bayer CropScience LP. 7 p.
47871500
Bayer CropScience, LP (2009) Submission of Product Chemistry and Toxicity Data in Support of the Application for Registration of Cry2Ae. Transmittal of 4 Studies.
47871501
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Product Analysis Studies as Outlined in 40CFR 158.2120-Cry2Ae. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47871502
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Toxicology Studies to Test for Health Effects as Outlined in 40CFR 158.2140-Cry2Ae. Unpublished study prepared by Bayer CropScience, LP. 9 p.
47871503
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Nontarget and Environmental Fate Studies to Test for Ecological Effects as Outlined in 40CFR 158.3150-Cry2Ae. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47871504
Hunst, P. (2009) Bridging Data from TwinLink(TM) Cotton Avian Nutritional Study to Parental Events T304-40 and GHB119. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47871600
Bayer CropScience, LP (2009) Submission of Product Chemistry and Toxicity Data in Support of the Application for Registration of BCS TwinLink(TM) Cotton Event T304-40xGHB 119 Cotton. Transmittal of 6 Studies.
47871601
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Product Analysis Studies. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47871602
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Toxicology Studies to Test for Health Effects as Outlined in TwinLink. Unpublished study prepared by Bayer CropScience. 9 p.
47871603
Coats, I. (2009) Request for Waivers from the Requirement to Conduct Certain Nontarget and Environmental Fate Studies to Test for Ecological Effects - TwinLink. Unpublished studies prepared by Bayer CropScience. 7 p.
47871604
Currier, T. (2009) Protein Expression Analysis for Pat/bar, Cry1Ab and Cry2Ae Proteins in Whole TwinLink(TM) Plants Grown in the Field. Project Number: CP09Q005. Unpublished study prepared by Bayer CropScience, LP. 28 p.
47871605
Hunst, P. (2009) Investigation of Synergistic Effects of the Cry1Ab and Cry2Ae Proteins expressed in TwinLink(TM) Cotton (BCS-GH004-7 x BCS-GH005-8) on Target Insect Pests Using Leaf Bioassay. Unpublished study prepared by Bayer CropScience, LP. 8 p.
47871606
Coats, I. (2009) Supplement to MRID # 47640014: Insect Resistance Management Plan: TwinLink(TM) Cotton. Unpublished study prepared by Bayer CropScience, LP. 7 p.
47903100
Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS TwinLink(TM) Cotton Event T304-40 x GHB 119 Cotton. Transmittal of 1 Study.
47903101
Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian Oral Toxicity Test: TwinLink. Unpublished study prepared by Bayer CropScience LP. 16 p.
47903300
Bayer CropScience LP (2009) Submission of Toxicity Data in Support of the Application for Registration of BCS CRY2AE Cotton Event GHB119. Transmittal of 1 Study.
47903301
Hunst, P. (2009) Request for Waiver from the Requirement to Conduct an Avian Oral Toxicity Test: TwinLink. Unpublished study prepared by Bayer CropScience LP. 16 p.
47928800
Bayer CropScience, LP-BioScience. (2009) Submission of Efficacy Data in Support of the Registration of BSC TwinLink(TM) Cotton Event T304-40 X GHB 119 Cotton. Transmittal of 1 Study.
47928801
Coats, I. (2009) Addendum to Product Chemistry for TwinLink: Structural and Functional Equivalence of Cry1Ab and Cry2Ae and PAT/bar Proteins Produced in Bacillus thuringiensis (Bt) and Escherichia coli to Cry1Ab, Cry2Aa and PAT/Bar in TwinLink(TM) Cotton, Gossypium hirsutum, USA, 2007: Final Report. Project Number: CY07B006. Unpublished study prepared by Bayer CropScience, LP. 7 p.
48177500
Bayer CropScience LP (2010) Submission of Toxicity Data in Support of the Applications for Registration of BCS TwinLink(TM) Cotton Event T304-40 X GHB 119 Cotton, BCS Cry 1Ab Cotton Event T304-40, and BCS Cry2Ae Cotton Event GHB119 and the Petition for Tolerance of Cry2Ae. Transmittal of 1 Study.
48177501
Stafford, J. (2010) Northern Bobwhite (Colinus virginianus) Dietary Toxicity Test (LC50) with GHB119 (Cry2AE) Cottonseed Meal. Project Number: 13798/4130, TX44L001. Unpublished study prepared by Springborn Smithers Laboratories. 60 p.
48427900
Bayer CropScience (2011) Submission of Efficacy Data in Support of the Experimental Use of T304-40 x GHB119 x COT102 x COT67B in Cotton. Transmittal of 1 Study.
48427901
Robinson, T. (2011) Cry1Ab, Cry2Ae, PAT/bar, FLCry1Ab, Vip3Aa, APH4 Levels in Selected Tissues of the Combined Trait Product T304-40 x GHB119 x COT102 x COT67B (BCS-GHOO4-7 x BCS- GH005-8 x SYN-IR1O2-7 x SYN-IRO67-1). Project Number: 10/BM45L001. Unpublished study prepared by Bayer CropScience, LP. 22 p.
48471900
Bayer CropScience (2011) Submission of Product Chemistry Data in Support of the Experimental Use of Cry2Ae (GEM2) in Cotton. Transmittal of 1 Study.
48471901
Currier, T. (2011) Analysis to Determine if the Cry2Ae (GEM2) Protein from Cotton Leaves is Glycosylated: Final Report. Project Number: BK05B003. Unpublished study prepared by Bayer CropScience. 32 p.
48480000
Bayer CropScience LP (2011) Submission of Product Chemistry and Efficacy Data in Support of the Applications for Registration of BCS Cry1Ab Cotton Event T304-40, BCS Cry2Ae Cotton Event GHB119 and BCS TwinLink(TM) Cotton Event T304-40 x GHB 119 Cotton, and the Petition for Tolerance of Cry2Ae. Transmittal of 11 Studies.
48480001
Habex, V.; Verhaeghe, S. (2008) Detailed Insert Characterization of Gossypium hirsutum Transformation Event GHB119 by Southern Blot Analysis. Project Number: BBS07/009, M/308404/02/1. Unpublished study prepared by Bayer BioScience N.V. 43 p.
48480002
Moens, S. (2008) Confirmation of the Absence of Vector Backbone Sequences in Gossypium hirsutum Transformation Event GHB119. Project Number: BBS06/007, M/311127/02/1. Unpublished study prepared by Bayer BioScience N.V. 40 p.
48480003
Moens, S.; Criel, I. (2008) Detailed Insert Characterization of Gossypium hirsutum Transformation Event T304-40. Project Number: BBS07/014, M/311245/03/1. Unpublished study prepared by Bayer BioScience N.V. 62 p.
48480004
Habex, V.; Van Herck, H. (2007) Confirmation of the Absence/Presence of Vector Backbone Sequences in Gossypium hirsutum Transformation Event T304-40: Final Report. Project Number: BBS05/003, M/308400/02/1. Unpublished study prepared by Bayer BioScience N.V. 41 p.
48480005
Currier, T. (2011) Characterization of Cry1Ab and (Inert Ingredient) Proteins Produced in Event T304-40 Cotton, Gossypium hirsutum USA, 2011. Project Number: 10/BM99L508, M/406631/01/1. Unpublished study prepared by Bayer CropScience LP. 31 p.
48480006
Moens, S. (2011) Characterization of Cry2Ae and (Inert Ingredient) Proteins Expressed in Gossypium hirsutum Transformation Event GHB119. Project Number: BIO2/005/PROTEQUL/512, M/406615/02/1. Unpublished study prepared by Bayer BioScience N.V. 35 p.
48480007
Currier, T. (2011) Structural Stability Analysis of Gossypium hirsutum Combined Event TwinLink(TM) X GlyTol (GHB614): Final Report. Project Number: CP08Q012/2. Unpublished study prepared by Bayer CropScience LP. 34 p.
48480008
Currier, T.; Harbin, A. (2011) Protein Expression Analysis for GlyTol x TwinLink(TM) (GHB614 x T304-40 x GHB119) Cotton Grown in the Field, USA 2010. Project Number: 10BM99L440B, M/406245/01/1. Unpublished study prepared by Bayer CropScience LP. 125 p.
48480009
Bushey, D.; Jansens, S.; Becker, A. (2010) Insect Bioassay of Plant Material Expressing Cotton Events T304-40, GHB119, and TwinLink(TM) (T304-40xGHB119). Project Number: BIO2/005, INSECT/BIOASSAY/FROM/MONHEIM. Unpublished study prepared by Bayer CropScience. 22 p.
48480010
Mullins, W. (2011) High Dose Study: Estimating the Mortality of Heliothis virescens in a Mississippi Cotton Field on Caged Plants of T304-40 x GHB119 (TwinLink) Cotton (BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Events T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Unpublished study prepared by Bayer CropScience LP. 16 p.
48480011
Dennehy, T. (2011) High Dose Study: Estimating the Mortality of Heliothis virescens in a Greenhouse on Plants of T304-40 x GHB119 (TwinLink) Cotton (BCS-GHOO4-7 x BCS-GHOO5-8) and Parental Event Plants T304-40 (Cry1Ab) and GHB119 (Cry2Ae). Project Number: BA44Y001. Unpublished study prepared by Bayer CropScience LP. 16 p.
48495100
Bayer CropScience (2011) Submission of Efficacy Data in Support of the Application for Registration of the TwinLink. Transmittal of 1 Study.
48495101
Dennehy, T. (2011) A Risk Assessment of TwinLink(TM) Cotton and Tobacco Budworm (Heliothis virescens) in the Midsouth. Unpublished study prepared by Mississippi State University. 15 p.

Other References

Safe use  -  mode of operation

Ferré, J. and Van Rie, J. 2002. Biochemistry and genetics of insect resistance to Bacillus
thuringiensis. Ann. Rev. Entomol. 47:501-533.

Organization for Economic Co-operation and Development (OECD). 2007. Consensus document
on safety information on transgenic plants expressing Bacillus thuringiensis-derived
insect control protein. 109 pages.