June 8 - 10, 2004 Charge/Questions to the Panel
May 19, 2004
FIFRA SCIENTIFIC ADVISORY PANEL (SAP)
JUNE 8-10, 2004
FIFRA SAP WEB SITE http://www.epa.gov/scipoly/sap/
OPP Docket Telephone: (703) 305-5805
Docket Number: OPP-2004-0146
PRODUCT CHARACTERIZATION, HUMAN HEALTH RISK,
ECOLOGICAL RISK, AND INSECT RESISTANCE
MANAGEMENT FOR Bacillus thuringiensis (Bt) Cotton Products
CHARGE/QUESTIONS TO THE PANEL
WideStrike Cotton Questions
Product Characterization/Human Health Risk Assessment
1. The Agency examines the safety of proteins based on the source of the protein, the protein’s pesticidal mode of action, comparisons of the amino acid sequence to toxins and allergens and the results of acute oral toxicity testing. The company provided numerous mammalian oral toxicity studies to demonstrate the safety of the introduced Cry 1Ac and Cry1F protein insecticidal toxins. The toxins were tested both separately and in combination. The Agency believes tests with combinations of pure proteins may address possible synergistic interactions between introduced proteins. However, the Agency believes that unless there is an indication that the two proteins would interact, such as being parts of a binary toxin or attaching to the same receptor, there is little to justify testing the two proteins together when separate oral toxicity tests indicate a lack of toxicity for the individual proteins.
Does the Panel have additional comments on this position including identifying instances where it would be justified to require the toxicity testing of two proteins in combination?
2. When traits are introduced into crop plants using the transformation techniques of modern biotechnology or even traditional breeding, one of the areas of concern is the possibility of unintentional changes. There is a general difficulty in screening for these unforeseen changes since it is a conceptual leap to anticipate the unexpected. However, in general the approach has been to examine general performance of the new cultivars like agronomic performance and compositional analysis to detect unintentional effects. PIP products can be both transformed lines and the result of traditional breeding of two transformed lines to yield a new product with combined traits like WideStrike cotton. In both cases, the new PIP product must be registered just as other new combinations of pesticide active ingredients must be registered.
For PIP products resulting from traditionally bred transformed lines, under what circumstances, if any, would it be appropriate to examine agronomic performance and compositional analysis to provide a screen for unintentional changes in the crop? Please describe other ways EPA might consider screening for potential unintentional changes in a crop.
Ecological Risk Assessment
1. WideStrike cotton is a product expressing pyramided Cry1F and Cry1Ac Bt proteins. The submitted non-target effects studies examined the effects of the Cry1F and Cry1Ac proteins separately and in combination to detect any synergistic effects on non-target wildlife. No synergistic effects or increase in non-target host range were seen as a result of combining these two proteins in the same product.
The Panel is requested to comment on the need for non-target hazard data development on the combinations of Cry proteins being considered for registration when data on the effects of the individual Cry proteins are readily available and show no adverse effects.
2. The weight of evidence from the reviewed data indicates that there will not be a hazard to wildlife from the commercialization of WideStrike cotton. Although the Bt proteins expressed by WideStrike are known to affect only lepidopteran insect species, the Agency evaluated studies of potential effects on a wide variety of non-target organisms that might be exposed to the Cry1F and Cry1Ac protein, i.e., wild mammals, birds, invertebrates, and aquatic species. EPA concluded that aquatic and terrestrial wildlife was not likely to be harmed and that WideStrike cotton was not likely to threaten the long-term survival of any non-target wildlife populations.
The Panel is requested to comment on the Agency's analysis of the currently available data on the potential impacts of WideStrike cotton on non-target species.
3. The Agency has sufficient information to conclude that there is no hazard from the proposed uses of WideStrike™ cotton to non-target wildlife, aquatic and soil organisms. However, the Agency is requesting additional, primarily long term effects data that were recommended by previous Panels for PIP corn. The supplementary studies would provide additional weight to support the Agency's conclusions.
The Panel is asked to comment on (a) the scientific value of the proposed additional studies that are identified at the end of the Environmental Assessment section, including avian chronic exposure testing and multi-year field and soil persistence/terrestrial expression studies, and (b) the applicability of these data to PIP cotton.
Insect Resistance Management
1. Dose. Three methods (two laboratory and one field) outlined by USEPA’s Scientific Advisory Panel (1998) were used to demonstrate that WideStrike cotton expresses a high dose of Cry1Ac and Cry1F against tobacco budworm (Heliothis virescens, TBW). Dow AgroSciences (Dow) employed one laboratory-based and one field-based method to demonstrate that WideStrike cotton has a high dose (Cry1Ac only, Cry1F is non-toxic) against pink bollworm (Pectinophora gossypiella, PBW) Results of two field studies indicate that WideStrike cotton produces a moderate dose against cotton bollworm (Helicoverpa zea, CBW), but a very high level of control (94%). The Agency concluded that WideStrike cotton expresses a high dose of Cry1F and Cry1Ac against TBW (Cry1Ac alone expresses a high dose and Cry1F a nearly high dose); a moderate dose of Cry1F and Cry1Ac against CBW, and a high dose of Cry1Ac against pink bollworm.
The Agency asks the SAP to comment on the Agency’s analysis of dose for TBW, CBW, and PBW, the likelihood that resistance will be inherited as a recessive trait, and its impact on insect resistance management for WideStrike cotton.
2. Cross-resistance. Resistance to Bt proteins can occur through several different mechanisms. Alteration of binding receptors has been the most common mechanism observed. The binding patterns of the Cry1F and Cry1Ac proteins in CBW and TBW indicate there are shared and unique binding sites. In TBW, Cry1Ac binds to at least three receptors, while Cry1F binds to at least two, only one of which binds Cry1Ac. In CBW, Cry1Ac and Cry1F each bind to at least four receptors, of which two are shared. For CBW, approximately 60% of Cry1Ac binding is to receptors that also bind Cry1F, and the remaining 40% of Cry1Ac binding is to receptors that do not bind Cry1F. Incomplete shared binding is expected to lead to incomplete cross-resistance when resistance is mediated by receptor changes. Thus, a mutation in a gene that codes for a receptor that binds both insecticidal control proteins (ICPs) will not prevent all binding of either ICP and thus alone will not allow high survival of the insect bearing even two copies of it, on WideStrike (Cry1F/Cry1Ac) cotton plants.
The Agency asks the SAP to comment on EPA’s conclusion that incomplete shared binding of Cry1Ac and Cry1F receptors, in TBW and CBW, is expected to lead to incomplete cross-resistance and thus the likelihood of enhanced survival on WideStrike cotton is expected to be small. Please comment on EPA’s conclusion that resistance is more likely to be associated with receptor binding modifications rather than other mechanisms of resistance such as detoxification in the midgut lumen by proteases that cleave the insecticidal control protein(s), metabolic adaptations, protease inhibition, gut recovery, and behavioral adaptations.
3. CBW modeling. Dow’s CBW modeling efforts show that EPA can have high confidence that there will not be a significant change in population fitness of CBW on WideStrike cotton in a 15-year time horizon even without a high dose for either Cry1Ac or Cry1F and incomplete cross-resistance (20 to 60% maximum shared binding). Market share analysis of WideStrike cotton versus other Bt cotton products had little effect on the rate at which CBW may adapt in either the North Carolina or Mississippi Delta agroecosystem. Refuge size, whether sprayed or unsprayed, had no significant impact on CBW population fitness on WideStrike cotton after 15 years. In the Delta, the immigrating non-selected population from alternate hosts further reduces the local rate of adaptation. The local structured refuge only supplies a small proportion of the non-selected insects in the Delta. The availability of CBW alternate hosts, coupled with a non- Bt cotton refuge are additional levels of assurance for WideStrike cotton product durability. Additional empirical information is needed on the function and effectiveness of alternate hosts on the rate of CBW adaption.
The Agency asks the SAP to comment on the predictions made by the DAS CBW model, i.e., the likelihood that the population fitness of CBW on WideStrike cotton in a 15-year time horizon will remain unchanged, even without a high dose for either Cry1Ac or Cry1F and incomplete cross-resistance (60% of Cry1Ac binds to the Cry1F receptor).
4. TBW modeling. For TBW, durability is expected to be greater than that predicted using the TBW model by Peck et al. (1999) where the worst case (structured refuge is moved each year) is 17 years. TBW exhibits similar patterns in binding studies as does CBW and WideStrike cotton expresses a high dose against TBW. The Cry1Ac component alone is a high dose and the Cry1F component alone is not quite a high dose.
The Agency asks the SAP to comment on the relative WideStrike cotton durability against TBW using the Peck et al. (1999) model.
5. Alternate hosts. Dow utilizes its CBW model that simulates two agroecosystems that consist of CBW crops hosts soybean, maize, and cotton in varying amounts, three insecticidal control proteins (Cry1Ac, Cry1F, and Cry2Ab), and three protein receptors. Dow also uses the HOSTS data base, and carbon isotope work by Gould et al. (2002) to support the use of CBW alternate hosts as an effective means of reducing the population-wide selection pressure to the two ICPs expressed in WideStrike cotton (metapopulation dynamics effects). To support the effectiveness of alternate hosts as natural refugia, data are needed on the larval and adult production of CBW on each alternate host for each generation relative to cotton and WideStrike cotton and the spatial scale and source of moth production.
The Agency asks the SAP to comment on:
a) the sufficiency of the WideStrike cotton database to address the issue of CBW alternate hosts as natural refugia, and,
b) whether additional data are needed on the larval and adult production of CBW on each alternate host for each generation relative to cotton and WideStrike cotton and the spatial scale and source of moth production to confirm the effectiveness of CBW alternate hosts as natural refugia.
6. IRM Plan. The WideStrike cotton IRM plan has the following proposed refuge requirements:
a. 5% external unsprayed refuge option. Five percent of the cotton fields must be planted to non-Bt cotton and not be treated with any lepidopteran-control technology. The refuge must be at least 150 ft. wide (preferably 300 ft.) and within ½ mile (preferably adjacent or within 1/4 mile or closer) of the Bt cotton.
b. 20% external sprayable refuge option. Twenty percent of the cotton fields must be planted to non-Bt cotton and may be treated with lepidopteran-active insecticides (or other control technology) except for microbial Bt formulations. The refuge must be within 1 mile (preferably within ½ mile or closer) of the Bt cotton fields.
c. 5% embedded refuge option for TBW and CBW. Five percent of a cotton field (or fields) must be planted with non-Bt cotton as a block within a single field, at least 150 ft. wide (preferably 300 ft. wide) or single-field blocks within a one mile squared field unit. The refuge
may be treated with lepidopteran-active insecticides (or other control technology) only if the entire field or field unit is treated at the same time.
d. Embedded (in-field strip) refuge option for PBW. One single row of a non-Bt cotton variety must be planted for every 6 to 10 rows of Bt cotton. This can be treated with lepidopteran-active insecticides (or other control technology) only if the entire field is treated at the same time.
e. Community refuge option. Farmers can combine neighboring fields within a one-mile squared field unit that act as a 20% sprayable refuge or the 5% unsprayed refuge. Participants in the community refuge option must have a community refuge coordinator, and appropriate documentation is required. It also includes the requirements for annual resistance monitoring, annual compliance assurance program, grower education, remedial action plans, and annual reporting. Any plan that focuses on TBW, CBW, and PBW should be adequate to maintain susceptibility in secondary pests, such as fall armyworm, beet armyworm, southern armyworm, cabbage looper, and soybean looper. A market mix of different Bt cottons and other control technologies further reduces the expected selection pressure for resistance from the Cry1F and Cry1Ac proteins expressed in WideStrike cotton.
The Agency asks the SAP to comment on the scientific data available to support the proposed IRM plan and whether that data support a delay in resistance of TBW, CBW, and PBW resistance to the Cry1F and Cry1Ac proteins expressed in WideStrike cotton for at least 15 years.
Bollgard and Bollgard II cotton IRM: Cotton Bollworm IRM Questions
As a condition of the Bollgard and Bollgard II registrations, EPA required that the Monsanto Company conduct CBW alternate host research studies and pyrethroid overspray studies to support the adequacy of the 5% external, unsprayed, structured refuge. In addition, EPA required that the Monsanto Company conduct research on the north-south movement, i.e., reverse migration, of CBW and its impact on Bt corn and cotton insect resistance management.
1. North-south movement. Based on the modeling studies submitted using the data in Gould et al. (2002), CBW (also called corn earworm in corn) reverse migration has no significant impact (0.05<P) on CBW adaptation to Bt corn and cotton.
The Agency requests that the SAP comment on whether CBW reverse migration is expected to have any significant impact on CBW adaptation to Bt crops.
2. Pyrethroid oversprays. Pyrethroid oversprays in Bollgard cotton fields will increase the level of control of CBW, delay the evolution of resistance, and increase the relative effectiveness of the 5% external, unsprayed, structured refuge. These findings support the general predictions of the Gustafson et al. (2001/2004) model. Pyrethroid sprays on Bollgard II plots do not provide a statistically significant difference in reduction of CBW infestation or damage from untreated Bollgard II cotton fields or from treated Bollgard cotton fields, and should not be included as a parameter in the Gustafson et al. (2004) model.
a. The Agency requests that the SAP comment on whether pyrethroid oversprays in Bollgard cotton fields are likely to increase the level of control of CBW, delay the evolution of resistance, and increase the relative effectiveness of the 5% external, unsprayed, structured refuge.
b. The Agency also requests that the SAP comment on EPA’s recommendation that pyrethroid oversprays not be included as a parameter in the Gustafson et al. (2004) model for Bollgard II.
c. Marcus et al. (2004) found that CBW larvae (late instars) in North Carolina Bollgard plots were half as susceptible to Cry1Ac (i.e., more tolerant) as were populations from non-Bollgard cotton survivors in the F1 generation.
The Agency requests the SAP comment on whether the cotton bollworm larvae coming from Bollgard fields are more tolerant to the Cry1Ac protein than those larvae coming from the non-Bollgard fields. What, if any, additional genetic work should be conducted to better understand the nature of this Cry1Ac tolerance.
d. The Agency requests the SAP to comment on the value of using a Cry1Ac-resistant CBW colony to investigate the genetic basis for CBW survival on Bollgard cotton.
3. Alternate hosts. Based on the two-year, studies in five states, both C3 and C4 alternate hosts serve as unstructured refugia. Data show that CBW moths are produced on alternate hosts throughout the landscape (spatial scale is greater than 10 miles) in sufficient numbers throughout the cotton growing season to mate with any putative resistant CBW moths emerging in Bollgard or Bollgard II cotton fields and dilute resistance. That is, the susceptible CBW moths coming from alternate hosts will reduce the intensity of Cry1Ac and Cry2Ab2 resistance selection in CBW and lower the likelihood of resistance evolution. The contribution of susceptible CBW adults from alternate hosts is greater than that from the 5% external, unsprayed, structured non-Bt cotton refuge. Despite the limitations EPA has identified associated with the Gustafson et al. (2001/2004) model, the CBW alternate host data support the model’s predictions that alternate hosts will substantially delay resistance.
a. Based on the larval productivity analyses, adult productivity analyses, and satellite imaging analysis, the Agency asks the SAP to comment on the relative contribution of the C3 and C4 alternate hosts as unstructured refugia to dilute CBW resistance.
b. Based on the data, the Agency also asks the SAP to comment on the spatial and temporal scale across the landscape, e.g., 1 mile, 10 mile etc., in which CBW adult production should be evaluated.
c. EPA concludes that “effective refuge size” should be a weighted average of the proportion of moths coming from each alternate host for each CBW generation (5 to 6 generations) in each cotton production system (geography).
The Agency asks the SAP to comment on how to quantitatively or semi-quantitatively calculate “effective refuge size” locally and regionally using available data (see above).
4. Gustafson et al. CBW model. Monsanto modified Caprio’s (1998a) two-patch, deterministic, non-random, population genetics model (publically available) to create a new CBW model, Gustafson et al. (2004, originally submitted to the Agency in September 2001 as part of the Bt Crops Reassessment) that included alternate hosts and synthetic pyrethroid oversprays as parameters. Sensitivity analyses showed that the model output (years to resistance) was sensitive to both of these parameters. Gustafson et al. (2004) have calculated “effective refuge size” as the sum of the total acres by county represented by the four alternate crop hosts – corn, sorghum, peanuts, and soybeans, and wild hosts (defaulted as 10% of the cotton acreage) as a percent of cotton acres. This model predicts that the 5% external, unsprayed, structured refuge option is adequately protective to delay CBW resistance if effective refuge size (alternate hosts) and typical use practices for Bollgard cotton, i.e., synthetic pyrethroid oversprays, are included. When this model was submitted to the Agency in 2001, empirical data to support the use of alternate hosts and synthetic pyrethroid were lacking.
a. The Agency asks the SAP to comment on the “effective refuge size” calculation. Does the SAP agree with the Agency’s conclusion that “effective refuge size” is a weighted average of the proportion of moths coming from each alternate host for each CBW generation (5 to 6 generations) in each cotton production system (geography)?
b. The Agency requests the SAP to comment on the strengths and weaknesses of the Gustafson et al. (2004) model and its utility with regard to the effective contribution of alternate hosts as natural refuge per generation. How would the model output be altered if the calculation of “effective refuge size” is changed (see a. above). What are the SAP’s recommendations for refining the Gustafson et al. (2004) CBW resistance management model or using a different CBW resistance management model to more appropriately consider the spatial and temporal dynamics of CBW utilization of alternative hosts by generation based on the data in Head and Voth (2004)?
c. The Agency requests the SAP to comment on validity of using the average pyrethroid efficacy value against CBW based on all the field studies conducted in all four states (North Carolina, Louisiana, Mississippi, and South Carolina) as the parameter value in the Gustafson et al. (2004) model rather than just the Brickle et al. (2001) data from South Carolina.