October 20, 2010

MEMORANDUM

SUBJECT: Review of Insect Resistance Ma nagement (IRM) considerations with a proposed reduced refuge for Bt corn event 1507 x MON 810

in the Corn Belt. EPA Reg No. 29964-7. MRID# 480056-01, -02, -03, -04. PC Codes: 006481, 006490. Decision#: 429187.

DP Barcodes: 375491.

TO: Ann Sibold, Regulatory Action Leader

Microbial Pesticides Branch Biopesticides and Pollution Prevention Division (7511P)

FROM: Alan Reynolds, Entomologist /s/

Microbial Pesticides Branch Biopesticides and Pollution Prevention Division (7511P)

PEER

REVIEW: Jeannette Martinez, Ecologist /s/

Microbial Pesticides Branch Biopesticides and Pollution Prevention Division (7511P)

Action Requested

BPPD 1 has been asked to review Insect Re sistance Management (IRM) data and information in support of an amendment to the Bt corn pyramid event 1507 x MON 810 (EPA Reg No. 29964-7). The proposed amendment requests a reduced 5% refuge for lepidopteran corn pests in the Corn Belt. Registration of 1507 x MON 810 was originally approved with a 20% refuge in the Corn Be lt. Supporting data are contained in four study reports (MRID# 480056-01, -02, -03, -04).

Conclusions and Recommendations

1) Pioneer has provided adequate data to scientifically support lowering the required refuge for 1507 x MON 810 corn (EPA Reg N o. 29964-7) from 20% to 5% in the Corn

1 The use of BPPD in this review refers to the B PPD IRM Team consisting of Alan Reynolds and Jeannette

Martinez- 2 - Belt. Information submitted by Pioneer included data on dose expression (previously reviewed in BPPD 2010a), cross resistance po tential for Cry1Ab and Cry1F, and simulation modeling for two major target pest s, European corn borer and southwestern

corn borer. These data demonstrate that the durability of 1507 x MON 810 with a 5% refuge is greater than either single to xin 1507 (Cry1F) or MON 810 (Cry1Ab) with the standard 20% refuge.

2) Data generated by Pioneer (MRID# 480056- 01) and published literature indicate that Cry1F and Cry1Ab have low cross resistan ce potential in ECB and likely SWCB and corn earworm (CEW). Pioneer employed a ra nge of approaches to investigate Cry1F - Cry1Ab cross resistance incl uding structural comparis ons, midgut (receptor) binding assays, pore formation analysis, and tests wi th Cry1F-resistant colonies. These data largely show that Cry1Ab and Cry1F elicit toxic responses through separate pathways. Midgut binding studies identified several shared binding sites in each of the target which could indicate some degree of cross resist ance. On the other hand, experiments with Cry1F-resistant ECB showed no survival on Cr y1Ab. The potential for low level cross resistance between Cry1F and Cry1Ab was a ddressed by exploring a range of values using a sensitivity analysis w ith the simulation model.

3) Pioneer’s simulation model showed that ev en with a smaller (5%) refuge, the 1507 x MON 810 pyramid is substantially more dur able than either 1507 (Cry1F) or MON 810 (Cry1Ab) alone with the standard 20% re fuge. For ECB, 1507 x MON 810 was more than four times as durable as MON 810 (136 years vs. 33 years) and 12 times as durable as 1507 (136 years vs. 11 years) using benchmark model parameters. For SWCB (with benchmark parameters), durability of the pyramid was seven times as durable as either 1507 or MON 810 alone (173 years vs. 22 years). “Worst case” simulations with highly conservative assumptions still showed that 1507 x MON 810 with a reduced refuge is predicted to be more durable for each target pest than either of the single toxin PIPs.

4) CEW simulations were not conducted for th e Corn Belt refuge proposal. BPPD agrees with Pioneer that resistance is unlikely for th is pest due to: 1) less prominence in the Corn Belt; 2) no overwintering in the Corn Belt; and 3) highly polyphagous host utilization that likely provides some natural refuge.

5) If the refuge proposal is approved, 1507 x MON 810 corn will have a different refuge requirement than many of the other registered Bt corn products that have a 20% refuge. Therefore, existing grower education and co mpliance will need to be modified (or developed) to accommodate the different re fuge size. BPPD supports the educational measures proposed by Pioneer, but also recommends increased emphasis on information supplied to growers, who must manage th e refuge requirements. It is further recommended that Pioneer submit revised copi es of their grower use guide to EPA to verify that the proper refuge instructions are included.

6) For compliance, BPPD recommends that Pioneer revise the established Compliance Assurance Program (CAP) for 1507 x MON 810 corn as it relates to the definition of a “significant” deviation with a 5% refuge. BPPD recommends that Pioneer remove the- 3 - “other” deviations category and define any amount of non-compliance with the reduced refuge as “significant.”

7) BPPD recommends that existing require ments for resistance monitoring, remedial action, and annual reporting be mainta ined for the amended 1507 x MON 810 registration.

Background

Bt corn event 1507 x MON 810 (EPA Reg. No. 29964-7) and 1507 x 59122 MON 810 (EPA Reg. No. 29964-8) were granted FIFRA section 3 registrations on February 24, 2010. The registrations are for pyramided pr oducts containing the Cry1F (event 1507) and Cry1Ab (event MON 810) toxins, which were derived by conventional breeding from Pioneer’s Herculex Cry1F product (E PA Reg. No. 29964-3) and Monsanto’s MON 810 Cry1Ab product (EPA Reg. No. 524-489). [Event 59122 expresses Cry34/35 for control of corn rootworm.] Both Cry1F a nd Cry1Ab are active against lepidopteran stalk boring pests, including European corn borer (ECB, Ostrinia nubilalis ), corn earworm (CEW, Helicoverpa zea ), and southwestern corn borer (SWCB, Diatraea grandiosella ). A number of other secondary lepidopte ran pests including fall armyworm (FAW, Spodoptera frugiperda ) and western bean cutworm (WBCW, Richia albicosta ) are also controlled or suppressed by Cry1F and/or Cry1Ab.

BPPD reviewed Insect Resistance Manage ment (IRM) considerations for 1507 x MON 810 (see BPPD 2010a) including data on dose (f ield efficacy and protein expression studies). Previous IRM assessments fo r Cry1F and Cry1Ab can be found in BPPD (2010b). At the time of application, Pioneer proposed to employ the refuge strategy employed for other Bt corn products: a 20% re fuge in the Corn Belt and a 50% refuge in cotton-growing areas in the southeastern U.S. The registration was subsequently granted with these refuge requirements.

Summary of Pioneer’s Amendment Request

Pioneer has submitted a proposal to reduce refuge s to 5% (from 20%) in the Corn Belt for 1507 x MON 810 corn only (29964-7). The othe r registered product containing 1507 and MON 810 (1507 x 59122 MON 810; EPA Reg. No. 29964-8) was not included with this amendment request. Supporting data for th e amendment are contained in four study

volumes:

MRID# 480056-01: Mode of Action and Cross Resistance of Cry1F and Cry1Ab in Ostrinia nubilalis (Hubner), Diatrea grandiosella (Dyer), and Helicoverpa zea (Boddie)

MRID# 480056-02: Predictive Model S upporting Reduced Block/Strip Lepidopteran Refuge for 1507 x MON810 Maize in the

Corn Belt- 4 - MRID# 480056-03: Trait Durability Plan Supporting Reduced Block/Strip Lepidopteran Refuge for 1507 x MON810 Maize in the

Corn Belt

MRID# 480056-04: Literature Referen ces Supporting Reduced Block/Strip Lepidopteran Refuge for 1507 x MON810 Maize in the

Corn Belt

Each of the first three studies listed above for Pioneer’s reduced refuge proposal is

addressed in the review below. Pioneer has also submitted a separate amendment to reduce refuge in cotton growing regions to 20% (from 50%) for two stacked products: 1507 x MON 810 (EPA Reg. No. 29964-7) and 1507 x 59122 MON 810 (EPA Reg. No. 29964-8). The cotton region amendment is review ed separately and is not addressed in

this memorandum.

Proposal for 5% Refuge in the Corn Belt

1) Mode of Action and Cross Resist ance of Cry1F and Cry1Ab (MRID# 480056-01)

Pioneer reasons that Cry1F and Cry1Ab in 1507 x MON 810 exert a “redundant killing” effect on the target insects in that both toxi ns act independently (w ithout cross resistance) and are expressed at high dose levels. This conclusion is supported by several lines of evidence including structural comparisons between the two protein toxins, midgut receptor binding characteristics, toxin activity (pore formation) analysis, and studies with Cry1F-resistant insect colonies.

a) Structural Analysis of Cry1F and Cry1Ab

Pioneer conducted a structural analysis of Cry1Ab and Cry1F by aligning the amino acid sequences of the proteins and comparing the domains (VectorNTI Advance software). Bt toxins are known to have three domains (DI, DII, DIII) that ar e involved in midgut receptor recognition and pore formation. Resu lts from the alignment analysis showed that Cry1Ab and Cry1F have <74% ami no acid protoxin sequence homology. Domain sequence similarities between the two toxins we re 68% (DI), 46% (DII), and 62% (DIII). Pioneer concluded that the low relative seque nce similarity supports the prediction that the two proteins utilize differe nt midgut receptor sites.

Pioneer also provided a struct ural comparison using three di mensional models of Cry1Ab and Cry1F (developed from the structural coor dinates of Cry1Aa). The two structural models were superimposed over each other so that differences in the domain loops could be visualized (shown in Figures 1 - 3 of Appendix 2 in MRID# 480056-01). These model comparisons show clear differences in the domains of the two proteins, particularly in DII and DIII. Domains II and III are known to be heavily involved in receptor recognition. Structural differences we re noted in the protein backbone as well as the side chains (known to be important for receptor binding).

b) Midgut receptor binding characteristics- 5 - Previous work with Cry1Ab and Cry1F bi nding properties (Hua et al. 2001) found a number of receptor sites in European corn borer (ECB) midguts. Although there were several shared binding sites, there was only weak competition between the two proteins

for these sites.

To further buttress the binding site work for ECB, Pioneer conducted new competitive binding and ligand blotting analysis for Cr y1Ab and Cry1F. The study also included corn earworm (CEW) and southwestern corn borer (SWCB), both of which had not yet been fully characterized for Cry1Ab/Cry1F bi nding interactions. Binding studies were conducted using Brush Border Membrane Vesicle (BBMV) prot ein obtained from dissected ECB, CEW, or SWCB midguts. Cry1Ab toxin (prepared using the methodology described in Hua et al. 2001) was labeled with Na 125 I for detection by radioactivity counters. Cry1F toxin used in the competitive binding testing (mature, trypsin activated; as with Cry1Ab) was mark ed using a fluorescent labeling technique (Alexa-Fluor) for detection by SDS-P AGE and an imaging device (Typhoon Trio Variable Mode Imager). Ligand blots were performed using BBMV proteins separated by SDS-PAGE and fixed on nitrocellulose me mbranes. Each membrane was incubated with either Cry1Ab or Cry1F toxin followe d by incubation with anti-Cry1Ac or antiCry1F antibodies. Secondary ( goat anti-rabbit) antibodies with fluorescent labeling were used for detection of the bound protein. West ern blots were also conducted using similar procedures to detect aminopeptidase-N (APN ) and alkaline phosphatase (ALP) proteins that could function as toxin receptors. Th e western blots included antiserum developed

from Heliothis virescens

, Manduca sexta , and Anopheles gambiae

.

Results from the Cry1F competition for Cry1Ab binding were similar for ECB, SWCB, and CEW (described in Appendi x 1 of MRID# 480056-01). In all cases, the presence of Cry1F did not inhibit Cry1Ab binding with BBMV proteins except at the highest concentrations of competitor. About 20% (ECB and CEW) to 40% (SWCB) competition (reduction in Cry1Ab binding) wa s observed at the higher co ncentrations of Cry1F. Conversely, homologous competition (Cry1Ab/ Cry1Ab) showed approximately 90% less

125 I-Cry1Ab binding at the maximum concentrations . A different pattern of results were observed with the reciprocal Cry1Ab comp etition for Cry1F (Alexa-Fluor marked) binding. Heterologous competition with Cry1Ab reduced Cry1F binding by approximately 70% (ECB) and 50% (SWCB). (The CEW binding tests were deemed unreliable because of high non-specific binding in the Cry1F/Cry1F homologous competition.) The high level of competition shown in the ECB and SWCB tests suggested that at least some Cry1F receptors are shared with Cry1Ab.

A second set of competitive binding studies were performed with Cry1F-resistant ECB (described in Appendix 2 in MRID# 480056-01) . Competitions were performed between labeled Cry1Ab and unlabeled Cry1Ab and Cry1F toxins. For susceptible ECB, the

results were similar to those described above with some competition (~50% reduction in Cry1Ab binding) recorded dur ing the Cry1Ab-Cry1F exposur e (suggesting shared and unshared binding sites). However, for Cry1F-re sistant ECB, the presence of Cry1F did not significantly inhibit Cry1A b binding indicating that the sh ared binding site was lost- 6 - (modified to no longer recognize Cry1F). Since homologous competition (Cry1AbCry1Ab) still existed with the resistant colony it is likely that an unshared Cry1Ab binding site remained viable.

Ligand blotting revealed a number of bindi ng proteins within ECB, SWCB, and CEW BBMVs for both Cry1Ab and Cry1F. A summar y of these binding sites is detailed in Table 1 below. Both Cry1Ab and Cry1F were shown to have distinct binding sites, though there were also a number of shared binding proteins in ECB and SWCB. Several

of these shared sites were identified as APN proteins (150 kDa in ECB; 180 kDa in SWCB).

Table 1. Cry1Ab and Cry1F Binding Proteins in ECB, SWCB, and CEW (modified from data provided in Table 1 of Appendix 1 in MRID# 480056-01)

Binding Proteins (kDa) 1

Cry1Ab Cry1F

ECB 25

32

40

50

100

150 (APN)

~210 25

35

60

150 (APN)

~270

SWCB 2 25 (ALP)

50 60 (ALP)

110

120

180 (APN)

210

>250 110

120 130 (APN)

180 (APN)

>250

CEW 3 60

90

130 150 (APN) ~20 (APN) 70 (ALP)

95 110 (APN)

1 Shared binding proteins are shown in bold. ANP = identified by western blot analysis as an aminopeptidase-N protein; ALP = identified by western blot analysis as an alkaline phosphatase protein.

2 SWCB ALP ( Anopheles gambiae ) protein was also detected at 40 kDa.

3 CEW APN proteins were also detected at 40 and 50 kDa ( Manduca sexta ) and 125 kDa ( Heliothis virescens ); ALP ( Anopheles gambiae ) protein detected at 45 kDa.

Given the apparently conflicting results between the Cry1Ab and Cry1F competitive binding studies, Pioneer suggested that the research presented “an incomplete picture of Cry1F (mode of action) in ECB.” The Cry1Ab - Cry1F binding competition showed strong Cry1F binding inhibiti on in the presence of Cry1Ab, though when Cry1F was introduced as a competitor to Cry1Ab bindi ng sites no competition was observed. One- 7 - possibility could be that Cry1F has fewe r binding receptors than Cry1Ab; however, Cry1F has shown to be highly effective (high dose) in registered corn hybrids. Another explanation could be that Cry1F has an additional unique binding site that was undetectable in the binding studies, possibly due to instability in the protein that is incompatible with BBMV processing. Pioneer noted that studies with Cry1Ab and Cry1F-resistant ECB colonies have shown little cross resistance.

Overall, Pioneer concluded that the bindi ng and blotting work provided evidence that Cry1Ab and Cry1F are compatible with a pyramiding toxin strategy. Ligand blotting showed that each toxin has multiple unique bi nding sites despite the presence of some shared binding proteins. Since multiple toxi n pathways (receptors) exists, there is the potential for “redundant killi ng” in each insect when Cry1Ab and Cry1F are deployed simultaneously in a pyramid. Cry1F competition assays for Cry1Ab binding sites

showed that two classes of receptors are found in ECB, SW CB, and CEW: one that is recognized by Cry1F and a second that is no t recognized by Cry1F. Pioneer indicated that the findings in their research supporte d similar conclusions reached by Hua et al. (2001).

c) Pore formation analys is (toxin activity)

Toxin activity by pore formation was assessed by using short circuit current measurements of dissected insect midguts. Th e assay is designed so that midgut tissue is mounted in a two-chambered arena (Ussing-st yle recording chamber) which is filled on both sides with a solution (the midgut tissue forms is positioned between in the opening between the two chambers). Electrodes are pl aced on either side of the midgut tissue to measure current (i.e., the movement of pota ssium ions across the “hemolymph” side to the “luminal” side). Bt toxins are added to the chamber facing the luminal side of the midgut. A reduction in the negative current across the midgut is an indication of pore formation by the toxin-receptor.

Pioneer conducted tests with dissected (Bt susceptible) ECB midguts and activated Cry1Ab and Cry1F toxins (Appendix 2 of MRID# 480056-01). Additional studies were performed on Cry1F-resistant ECB midguts. Pore formation was determined by the decay rate of the negative current. For susceptible ECB, both Cry1Ab (avg. 2.1 min) and Cry1F (avg. 6.6 min) formed pores quickly after introduction to the prepared midgut tissue. Conversely, there was only a small re sponse (indicating little pore formation) when Cry1F-resistant midguts were exposed to Cry1F in the recording chamber solution. Cry1F-resistant midguts were also exposed to Cry1Ab and showed rapid pore formation, comparable to the susceptible ECB mi dguts (4.4 min Cry1F-resistant vs. 2.1 min susceptible). Finally, Cry1F-resistant midgut s were exposed sequentially to Cry1F and then Cry1Ab. Initial Cry1F exposure had little effect (similar to the previous experiment) but the following Cry1Ab exposure resulted in quick and complete pore formation.

Based on these results, Pioneer concluded that the work provides further evidence of discrete modes of action for Cry1Ab and Cry1F . The results with Cry1F-resistant ECB showed that 1) Cry1F resistance is likely due to altered toxin receptors in the midgut, and- 8 - 2) Cry1Ab receptors were still functional ( pore forming) in Cry1F-resistant midguts. Sequential toxin studies with Cry1F-resistant ECB further de monstrated that there is no antagonism between the two toxins.

d) Cry1F-resistant insect experiments

In addition to the physiological and structur al assessments of Cry1Ab and Cry1F cross resistance, Pioneer conducted bioassays with Bt-resistant ECB. Two separate projects were conducted using several ECB coloni es resistant to the Cry1F toxin.

The first project was designed to assess the “redundant killing” of Cry1Ab and Cry1F in a pyramided transgenic corn environment (described in Appendix 3 in MRID# 480056- 01). A susceptible ECB colony (collected fr om Iowa and reared for seven generations) and Cry1F-resistant ECB colony were included in the testing. The resistant colony was originally collected from Hamilton Count y, Iowa in 2004 (held for 33 subsequent generations in culture) and ha s been characterized as having a recessive Cry1F resistance

gene.

Susceptible and resistant ECB neonates were exposed to corn leaf discs in growth chambers and assessed for mortality and growth inhibition. Treatments included 1507 (Cry1F only), MON 810 (Cry1Ab only), 1507 x MON 810 (pyramid of Cry1F and Cry1Ab), and non-Bt corn. After four days of exposure, the test arenas were checked for mortality (larvae that had not a dvanced past the first instar were considered dead) and any surviving larvae were weighed.

As shown in Table 2 below, both susceptib le and Cry1F-resistan t larvae exposed to isoline (non-Bt corn) showed relatively low mortality (21% susceptible and 14% resistant). On Cry1F (1507) leaf discs, th e resistant ECB also suffered low mortality (10.2%) compared to the susceptible colony (96 .9%). However, the mean weights of the surviving larvae from both colonies were similar (0.385 mg re sistant vs. 0.350 mg susceptible) and also comparable to the we ights from the isoline corn trials. This indicates that the susceptible larvae surviv ing Cry1F exposure likely experienced little fitness costs. On the other hand, complete mortality was observed for all ECB larvae (susceptible and resist ant colonies) that were expo sed to the Cry1Ab (MON 810) and pyramid (Cry1Ab x Cry1F) treatments. Ba sed on these results, the study authors concluded that a pyramid of Cry1F and Cr y1Ab provides redundant killing of ECB even for Cry1F-resistant larvae.

Table 2. Susceptibility of Cry1F-Resistant a nd Reference ECB Colonies to Cry1Ab and Cry1F in Single and Pyramid Treatments (tak en from data provided in Table 1 of Appendix 3 in MRID# 480056-01)

ECB colony Corn Disc Type % Mortality Avg. Larval Weight (mg) Susceptible Non-Bt 21.0 0.369 Cry1F-Resistant Non-Bt 14.0 0.389- 9 - ECB colony Corn Disc Type % Mortality Avg. Larval Weight (mg) Susceptible Cry1F 96.9 0.350 Cry1F-Resistant Cry1F 10.2 0.385 Susceptible Cry1Ab 100.0 no survivors Cry1F-Resistant Cry1Ab 100.0 no survivors Susceptible Cry1F x Cry1Ab 100.0 no survivors Cry1F-Resistant Cry1F x Cry1Ab 100.0 no survivors

The second experiment was conducted to fu rther genetically ch aracterize a Cry1Fresistant strain in terms of inheritance of resistance, sex-linkage, dominance, resistance loci, and cross resistance w ith Cry1Ab. Testing was conducte d using diet bioassays and susceptible and Cry1F-resistant ECB colonies . Resistant ECB (ori ginally obtained from collections in South Dakota, Minnesota, a nd Nebraska) were selected for at least 15 generations on Cry1F in the la boratory (reported to be the highest Bt resistance level achieved for ECB). Susceptible ECB (to bot h Cry1F and Cry1Ab) were collected from Iowa and maintained for at least 10 generati ons in culture. Reciprocal and backcrosses were also created from matings of the su sceptible and Cry1F-re sistant colonies.

Bioassays were conducted using diet overlay s of varying amounts (serial dilutions) of Cry1F or Cry1Ab toxin (untreated controls we re also tested). Neonate ECB larvae were exposed for a seven day period after which mo rtality was assessed. Dead larvae included

individuals that failed to a dvance past the first instar or weighed less than 0.1 mg. Reciprocal crosses between susceptible and re sistant ECB were used to investigate sex linkage and dominance while backcrosses (o f the reciprocal crosses with the Cry1Fresistance colony) were used to dete rmine the number of resistance loci.

Results from the experiment showed that the Cry1F-resistant ECB colony was highly tolerant of Cry1F (LC 50 >24,600 ng/cm 2 ) compared to the su sceptible colony (LC 50 = 0.43 ng/cm 2 ) giving a resistance ratio of >57,500 (Table 3). Reci procal crosses showed somewhat greater tolerance of Cry1F than the susceptible colonies (LC 50 = 1.34 ng/cm 2

for R ♂ x S ♀ , 1.25 ng/cm 2 for S ♂ x R ♀ , and 1.38 ng/cm 2 for the pooled cross). Given the similar responses among the F 1 reciprocal crosses, inherita nce appears to be autosomal and not sex-linked. For the susceptible (SS) , resistant (RR), and reciprocal crosses (F 1 pooled, RS) dominance was estimated (as a func tion of the survival of each genotype at each concentration tested). At the highe r bioassay concentrations (e.g, 8.0 and 40.0 ng/cm 2 ), dominance was low indicating recessive resistance. However, at lower concentrations (0.32 and 1.6 ng/cm 2 ), higher dominance values (close to 1.0) suggest almost complete dominance. A Chi Square te st performed on the mortality data for the backcross generation showed that the data followed a pattern expected for monogenic

resistance. This conclusion was further supported by the dose-response data which

showed about 50% survival for the backcross at the high test concentr ations (indicative of a population with 50% RR resulting from a monogenic backcross).

Cross resistance with Cry1Ab was assessed with diet bioassays of the susceptible and Cry1F-resistant colonies and reciprocal cr osses (Table 3). The susceptible colony- 10 - remained highly sensitive to Cry1Ab but the Cry1F-resistant colony showed some tolerance to Cry1Ab (resistan ce ratio = 10.5). However, th e reciprocal crosses were almost as sensitive to Cry1Ab as the sus ceptible colony (resistance ratios < 1.21). Additional testing with leaf discs showed that the Cry1F-resistant colony (tolerant of Cry1F leaf discs) was completely controll ed by leaf discs containing Cry1Ab or a pyramid of Cry1F and Cry1Ab.

Table 3. Responses of Susceptible and Cry1F-R esistant ECB Coloni es and Reciprocal Crosses to Cry1F and Cry1Ab Diet Bioassays (t aken from data provided in Tables 1 and 4 of Appendix 4 in MRID# 480056-01)

ECB Colony Cry1F Cry1Ab

LC 50 (ng/cm 2 ) Resistance

Ratio LC 50 (ng/cm 2 ) Resistance

Ratio

Susceptible (S) 0.43 -- 0.051 --
Cry1F Resistant (R) > 24,600 > 57,500 0.534 10.5
Reciprocal Cross -

R ♂ x S ♀ (F 1 ) 1.34 3.14 0.061 1.19

Reciprocal Cross -

S ♂ x R ♀ (F 1 ) 1.25 2.94 0.054 1.07

Reciprocal Cross - pooled (F 1 ) 1.38 3.24 0.062 1.21

Based on the data, the study author concl uded that Cry1F (1507) and Cry1Ab (MON 810) combined in a pyramid can extend tra it durability. Low level cross resistance observed in the Cry1Ab diet bioassays with the Cry1F-resistant strain ar e not likely to be significant enough to allow field survival (as ev idenced by the leaf disc tests). The Cry1F resistance in the selected colony was characterized as monogenic, non-sex linked (autosomal), and highly recessive.

BPPD Review - Mode of Action and Cross Resistance Potential of Cry1F and Cry1Ab

Overall, BPPD agrees with Pioneer that the s ubmitted data provide a scientific weight of evidence to support the conclusion that Cry1A b and Cry1F have low cross resistance potential and maintain independent modes of toxicity in ECB. Data provided for SWCB and CEW also suggest a similar conclusion for these insects. As such, Cry1Ab and Cry1F meet the lack of cro ss resistance criterion for simulta neous use in a pyramid (high toxin expression in the PIP is the other).

Pioneer conducted research in four major areas to test for Cry1F/Cry1Ab cross resistance: structural comparisons, midgut receptor bindin g characteristics, toxin activity (pore formation) analysis, and studies with Cry1F-resistant insect co lonies. These data largely show that Cry1Ab and Cry1F elicit toxic responses through separate pathways and support the findings of previ ously conducted studies. However, some of the work- 11 - suggests a low level of cross resistance between the toxins is possible, though it is unlikely to be significant enough to be field relevant.

Competitive midgut binding studies showed that Cry1Ab inhibited Cry1F binding by approximately 70% in ECB, which implies that at least some Cry1F receptors are shared with Cry1Ab. Ligand blotting confirmed the pr esence of several shared binding proteins (25 and 150 kDa) in ECB a lthough numerous other unshare d (unique) binding proteins were also identified. On the other hand, the presence of Cry1F in competitive binding assays did not inhibit Cry1Ab binding with BBM V proteins. A second test also showed that for a Cry1F-resistant ECB, Cry1F di d not significantly affect Cry1Ab binding indicating that an unshared Cry1Ab bindi ng site still remained viable.

The binding work conducted by Pioneer largel y confirmed previous research conducted by Hua et al. (2001). Ligand blotting in this study showed that both Cry1F and Cry1Ab recognize binding proteins of 154 and 220 kD as in ECB though Cry1Ab also recognized several other distinct proteins. In competitive binding experiments (surface plasmon resonance and radioligand competition) Cry1F did not impede Cry1Ab BBMV binding (except at the highest con centrations) although preincubation with Cry1Ab lowered Cry1F binding. Hua et al. (2001) concluded that Cry1F has low binding affinity for a Cry1Ab binding site and likely has a separate hi gh affinity site ( unrelated to Cry1Ab) responsible for toxicity in ECB. Separate research conducted by Pereira et al. (2010) found that Cry1F recognizes binding site s of 120, 200, and 250 kDa in ECB and that BBMV binding was not reduced with ECB selected for Cry1F resistance. The authors suggested that the mechanism for Cry1F resistan ce in ECB is specific and unrelated to Bt

toxin resistance mechanisms observed in other insects (e.g., reduced BBMV binding, increased activity of gut proteases). BPPD also notes that competitive binding studies conducted with a separate insect (diamondback moth, Plutella xylostella ) showed some competition between Cry1F and Cry1Ab for a common binding site, but also that Cry1Ab binds to separate sites independent of Cry1F (Granero et al. 1996).

Pioneer provided several explanations fo r the Cry1F/Cry1Ab bindi ng results including the possibility that Cry1F has fewer bindi ng receptors than Cr y1Ab (though Cry1F has shown to be efficacious in the field) or th at Cry1F has an additional unique binding site

that was undetectable in the studies. BPPD believes th ese studies do provide some

evidence of low level cross resistance thr ough the presence of shared binding sites in ECB BBMV. However, other submitted and pub lished data largely corroborate the view that both toxins likely recogni ze unique sites that cause most of toxicity in ECB.

Further evidence supporting l ack of Cry1F/Cry1Ab cross resistance was provided in studies conducted with Cry1F-resistant ECB. Pore formation analysis with Cry1Fresistant and susceptible ECB showed that ch annels formed readily in both susceptible and Cry1F resistant midguts after exposure to Cry1Ab. Exposure of the Cry1F-resistant midguts to Cry1F resulted in little pore formation, although subsequent introduction of Cry1Ab quickly induced pore formation. Since the creation midgut pores is thought to be responsible for the toxicity of Bt proteins, this work indicat es that Cry1Ab should still be effective against Cry1F-resistant ECB.- 12 - Susceptibility studies (leaf di sc and diet bioassays) with Cry1F-resistant ECB also showed that Cry1Ab still remained effective against these insects. Cry1Ab-expressing leaf discs (as a singl e toxin or pyramided with Cry1F) caused complete mortality to Cry1F-resistant larvae. In diet bioassays, Cry1F-resistant larvae were somewhat less susceptible to Cry1Ab compared to the uns elected control colony (resistance ratio = 10.5). This result could be due to the pres ence of shared Cry1F/Cry1Ab binding sites in ECB (as evidenced by the ligand blotting work). However, BPPD agrees with Pioneer that given the leaf disc results, this low le vel cross resistance is unlikely to be field relevant. The findings of Pioneer were larg ely similar to a separate investigation of Cry1Ab cross resistance with Cry1F-resistant ECB (Pereira et al. 2008). In this study, resistant ECB were nearly as susceptible to Cry1Ab as the control colony (resistance ratio = 1.5).

The overall analysis could have been buttr essed with reciprocal studies using Cry1Abresistant ECB. Resistant strains have been developed (see Siqueira et al. 2004, Crespo et al. 2009, and others), although it is unclear whethe r such colonies were (or are) available to Pioneer for testing. Pioneer reasoned th at such testing should not be undertaken because Cry1Ab resistance (polygenic) is di fferent than Cry1F resistance (monogenic) and Cry1Ab selection results in moderate re sistance (compared to the very high levels seen with Cry1F selection). Nonetheless, testing with Cr1Ab-resistant ECB could be insightful given the different patterns in binding inhibition that were shown in the Cry1AF/Cry1Ab competitive binding studies. Should Cry1Ab-resistant ECB be able to survive on Cry1F corn the benefits of the toxi n pyramid would be lost. In the work of Siqueira et al. (2004), some degree of Cry1F cross resistance was observed in two Cry1Ab-resistant ECB populations, though the leve ls were low (resistance ratios = 5.0 and 5.4)

Fewer data were submitted to investigate cross resistance potential with CEW and SWCB. Competitive binding and ligand blotting analyses were conducted and, like ECB, demonstrated distinct toxicity pa thways for CEW and SWCB. Some binding

inhibition between the toxins was observed for both insects and several shared binding sites were observed for SWCB. However, numerous unique (and unshared) binding sites were discovered in CEW and SWCB midguts (T able 1). Unlike the ECB work, separate

studies with these insects we re not conducted to assess pore formation or with resistant colonies. Yet given the binding analyses and structural differences between the toxins, it is likely that Cry1Ab and Cry1F will have a low cross re sistance potential in CEW and SWCB with little or no field relevance, similar to ECB.

2) Predictive Model Supporting Reduced Bl ock/Strip Lepidopteran Refuge for 1507 x MON810 (MRID# 480056-02)

To evaluate the durability of a reduc ed (5%) refuge for the 1507 x MON810 Cry1Ab/Cry1F pyramid, Pioneer created a deterministic, two gene simulation model. The model was designed for ECB and SWCB, but not CEW. Modeling was not undertaken for CEW due to: 1) lack of CEW prominence as a pest in the Corn Belt, 2)- 13 - low overwintering success of CEW in the Corn Belt, and 3) the polyphagous nature of CEW (potential natural refuge is available).

The primary objective of the modeling was to investigate the durability of a pyramid (Cry1F x Cry1Ab) with a 5% structured refuge relative to single trait PIPs (Cry1F or Cry1Ab) and a 20% structured refuge. Pion eer employed an approach similar to that taken by Roush (1998) in terms of paramete r assumptions. A comparison of the two modeling approaches was provided by Pi oneer and is described in Table 4.

Table 4. Comparison of Modeling Approaches Taken by Roush (1998) and Pioneer to Assess the Durability of Pyramided PIPs (t able reproduced verbat im from Table 1 of Pioneer’s submission, MRID# 480056-02)

Parameter Model Roush (1998) Pioneer (MRID 480056-02)
Model type Deterministic Number of Bt proteins

modeled One vs. Two

Initial frequency of

resistance alleles Single value based on

theoretical information Ranged based on empirical

and theoretical information

Fitness costs to resistance None Resistant homozygous

insects Unaffected by Bt plants

Egg survival Eggs laid on non-transg enic hosts survive equally well as those laid by resistant homo zygous insects on transgenic

hosts

Dose mortality 90-100% mortality for susceptible insects

70% mortality for

heterozygotes 95-100% mortality for susceptible insects

Heterozygote mortality

calculated from dominance values and susceptibility of homozygous resistant and susceptible insects

(The model simulates

recessive resistance to Cry1F and Cry1Ab so that heterozygotes respond the same as susceptible insects)

Cross resistance Modeled as full cross

resistance Varied (to allow for more flexibility and realism) Genetic linkage between

two resistance alleles Explored linkage Assumed no linkage

Mating Random throughout population- 14 - Parameter Model Roush (1998) Pioneer (MRID 480056-02)
Biological assumptions Two generations ECB or SWCB on maize/year; no natural refuge Bt protein exposure Occurs only for larvae Population growth Density independent Frequencies of genotypes Based on the Hardy-Weinberg equation

Additional parameters for Pioneer’s model are summarized in Table 5. Survival rates for the various genotypes (homozygous susceptible, heterozygous, and resistant) of ECB were determined using empirical data (Pioneer studies and peer revi ewed literature). For many of the potential genotypes, SWCB was assumed to respond similarly to ECB (in other cases, parameters were varied for SWCB). Homozygous resist ant individuals were

assumed to have 100% survival on their co rresponding single toxi n isolines. Both homozygous susceptible and heterozygous insect s were presumed to have 0% survival on Bt corn, with the exception of Cry1F resistan t heterozygotes whom were assumed to have 0-5% survival on Cry1F corn. Cross resi stance survival of Cry1F-resistant ECB on Cry1Ab was set to zero based on work with re sistant colonies (Pereira et al. 2008; see discussion in the previous section). For Cr y1Ab resistance, survival on Cry1F was set at 0.2 for ECB and 0.1 for SWCB; conservative le vels of cross resistance that Pioneer believes exceed those observed in Siqueira et al. (2004). Single toxi n Bt corn PIPs were modeled with a 20% structured refuge while the 1507 x MON 810 pyramid was assigned a 5% refuge (all refuges were a ssumed to be blocks or strips).

Table 5. Additional Modeling Parameters Employed in Pioneer’s Deterministic Model (taken from information provided in MRID# 480056-02)

Parameter Model Assumption Model duration 400 generations (200 years) Alleles A = Cry1Ab susceptible B = Cry1Ab resistant X = Cry1F susceptible Y = Cry1F resistant Initial Resistance allele frequency Cry1Ab (B): 0.001 (benchmark) Cry1F (Y): 0.01 (benchmark ECB), 0.001 (benchmark SWCB) Dominance (h) Cry1Ab, Cry1F: 0.01 (benchmark ECB) Cry1Ab, Cry1F: 0.02 (benchmark SWCB) Genotype survival AA, XX = 0.001 (ECB), 0.005 (SWCB) on Cry1Ab and Cry1F BB, YY = 1.0 on Cry1Ab and Cry1F AB, XY = (1-h)*S+h*s (on Cry1Ab and Cry1F)

Survival of genotypes on the Bt pyramid is a function of susceptible survival on Bt (S), susceptible survival on non-Bt (s), dominance (h),- 15 - Parameter Model Assumption cross resistance (CR), and survival of heterozygotes (Wrs) -- see table 3 in MRID#

480056-02 Cross resistance Cry1F to Cry1Ab: 0 Cry1Ab to Cry1F (ECB): 0.2 Cry1Ab to Cry1F (SWCB): 0.1 Refuge Single toxin Bt corn (1507 or MON 810): 20% Pyramid Bt Corn (1507 x MON 810): 5%

Refuges are structured a nd deployed as blocks or strips Definition of resistance Resistance allele frequency exceeding 0.5 Durability for each scenario is measured in years

to resistance

Pioneer’s modeling analyses included scenario s with “best,” “worst,” and “benchmark”

scenarios. Benchmark simulations were based upon empirical data and literature (described above and in the prev ious section) and were consider ed to be the most realistic parameters. The worst and best case scenarios incorporated the upper and lower limits of the parameter estimates, respectively. Additional sensitivity analyses, to determine the relative impact of certain variables on the model, we re conducted by varying one parameter while holding the others steady. Separate analyses were conducted for the cross resistance, dominance, susceptible surv ival rate, and initial resistance allele frequency parameters.

Prior use history (of Cry1Ab and Cry1F) was not specifically included in the models per se but was addressed by considering a range of values for initial resistance allele frequency. Selection pressure over time could result in an increase in resistance alleles within a population. Both Pioneer’s “worst case” and sensitivity analyses explored impacts of higher resistance allele fre quencies in ECB and SWCB on resistance development.

Results from the modeling are summarized in Table 6 below. For both ECB and SWCB, simulations conducted with the benchmark pa rameters predicted high durability of the Cru1F x Cry1Ab pyramid with a 5% refuge (136 years to resistance for ECB, 173 years for SWCB). Single toxin PIPs under the be nchmark parameters and a 20% refuge developed resistance in 33 years (Cry1Ab) and 11 years (Cry1F) for ECB and 22 years (Cry1Ab and Cry1F) for SWCB. Scenarios inco rporating best case parameters resulted in greater relative durability particularly for the Cry1F x Cry1Ab pyramid -- no resistance evolved among either ECB or SWCB w ithin 400 generations (200 years). Comparatively, resistance developed to Cr y1Ab in 63 years (ECB and SWCB) and to Cry1F within 20 years (ECB) and 63 years (SWCB). Resistance evolution was much quicker for simulations using the worst case assumptions. Durability for ECB was 9 years with the pyramid versus 8 years (Cry1A b) and 3 years (Cry1F) for the single toxin- 16 - varieties. For SWCB, resistance developed in 17 years on the pyramid and 6 years for both Cry1Ab and Cry1F corn.

Table 6. Model Output from Simulations of ECB and SWCB Adaptation to Single Toxin and Pyramided Cry1Ab and Cry1F Bt Corn (adapted from data provided in Tables 5 and 6 of MRID# 480056-02)

Insect Toxin (Bt corn type) Refuge Durability (years to resistance)

Benchmark Best case Worst case

ECB Cry1F x Cry1Ab 5% 136 >200 9 Cry1Ab 20% 33 63 8 Cry1F 20% 11 20 3

SWCB Cry1F x Cry1Ab 5% 173 >200 17 Cry1Ab 20% 22 63 6 Cry1F 20% 22 63 6

Pioneer emphasized that the model output (years to resistance) is not literal and should be used to make relative comparisons of dur ability. The company believes the modeling analysis is conservative give n 1) polyphagous feeding by the target pests, particularly ECB (not addressed in the model), 2) the a ssumption of no fitness costs for resistance (despite data suggesting such effects), and 3) conservative estimates of resistant insect survival on Bt plants (assumed to be 100%).

Sensitivity analyses were conducted for the E CB parameter estimates of cross resistance, survival of susceptible insects, initial resi stance allele frequency, and dominance (output summarized in Table 7). A range of high, me dium (i.e., benchmark), and low estimates were incorporated into simulations to estim ate the durability of a 5% refuge with the Cry1F x Cry1Ab pyramid. As shown in Tables 6 and 7, the medium/benchmark parameters established a baseline of 136 y ears durability with ECB. The model was sensitive to all four paramete rs as measured by the relativ e ranges of durability between the high and low estimates. Durability was greatl y reduced (to as little as 16 years) when higher estimates for initial resistance allele frequency and dominance were simulated. Conversely, resistance did not evolve under the low parameter es timates for initial resistance allele frequency and cross resist ance. The model was generally less sensitive

to the lower values used for survival of su sceptible insects and do minance (only slight increases in durability were noted).

Table 7. Sensitivity Analysis for ECB Modeling Parameters (taken from data provided in Tables 7, 8, 9, and 10 of MRID# 480056-02)

Parameter Value Durability of 5% refuge (years to resistance)

Survival Rate of Susceptible Insects 0.05 / 0.02 / 0.01 51 / 86 / 107

0.001 136

0.0001 141- 17 - Parameter Value Durability of 5% refuge (years to resistance)

Initial Resistance Allele Frequency Cry1Ab: 0.01 / 0.005 Cry1F: 0.1 / 0.05 16 / 31

Cry1Ab: 0.001

Cry1F: 0.01 136

Cry1Ab: 0.0005

Cry1F: 0.005 >200

Dominance Value of

Resistance Allele 0.2 / 0.1 / 0.05 16 / 36 / 70

0.01 136

0.005 / 0.001 145 / 151

Cross Resistance Cry1Ab → Cry1F: 0.5 / 0.4 / 0.3 Cry1F → Cry1Ab: 0.1 45 / 47 / 48

Cry1Ab → Cry1F: 0.2

Cry1F → Cry1Ab: 0 136

Cry1Ab → Cry1F: 0.1

Cry1F → Cry1Ab: 0 >200

BPPD Review - Predictive Modeling

Pioneer’s modeling shows that, under the pa rameter assumptions chosen, a pyramid of 1507 (Cry1F) and MON 810 (Cry1Ab) has superi or durability to single trait PIPs expressing Cry1Ab and Cry1F. In all sc enarios tested, 1507 x MON 810 with a 5% structured refuge was more durable then either 1507 or MON 810 with a 20% refuge. This was the case even with “worst case” estimates for some parameters that are not likely to be realistic for either ECB or SWCB.

Using the “benchmark” and “best case” parameter assumptions, the time to resistance for 1507 x MON 810 (with a 5% refuge) was far grea ter than the single trait products and a 20% refuge (see Table 6). For ECB, the pyram id was more than four times as durable as MON 810 (136 years vs. 33 years) and 12 times as durable as 1507 (11 years) with the benchmark parameters. The different initial resistance allele fre quency assumptions for Cry1Ab and Cry1F were probably responsible fo r the contrast in dur ability between the single toxin events. With SWCB, the benc hmark simulations showed that 1507 x MON

810 was more than 7 times as durable as ei ther 1507 or MON 810 alone (173 years vs. 22 years). In the best case scenarios, resistan ce to the pyramid failed to evolve to either ECB or SWCB during the model runs (400 generations).

“Worst case” analyses can also be useful to help determine the potential for resistance, particularly if there is uncertainty re garding the model input. With many IRM simulations, parameters such as initial resi stance allele frequency, dominance, and cross resistance typically include some degree of uncertainty and may be estimated based on “best guesses” for the insects and toxins. Pioneer’s worst case scenarios for ECB and SWCB addressed such questions by em ploying highly conservative assumptions,- 18 - particularly for Cry1Ab-Cry1F cr oss resistance, survival of su sceptibles on the Bt toxins,

and dominance of the resistance alleles. Thes e simulations resulted in greatly reduced durability for both the pyramid and single trait PIPs (compared to the benchmark runs -- see Table 6). Nevertheless, 1507 x MON 810 (with 5% refuge) remained comparatively more durable than 1507 or MON 810 (20% refu ge): for ECB, 9 years compared to 8 years (MON 810) and 3 years (1507); for SW CB, 17 years compared to 6 years (MON 810 and 1507). BPPD notes that the worst case simulations were likely unrealistic since they predicted very quick evolut ion of resistance to the single toxin PIPs (8 years or less). In fact, both Cry1Ab (registered since 1995) and Cry1F (register ed since 2001) have been in commercial use for much longer than the pr edicted time to resistance in the worst case modeling.

Output from Pioneer’s simula tions largely corroborates othe r modeling efforts that have examined pyramided approaches to resistan ce management. Such models, including Roush (1998), Zhao et al. (2003), and Gould et al. (2006), have shown that pyramids of two traits with independent modes of action (i.e., no cross resistance potential) and high activity (dose) can delay the e volution of resistance relative to single toxin deployments.

Lack of cross resistance has been addresse d by Pioneer in this submission (refer to the previous section); dose was previously asse ssed for the initial re gistration of 1507 x MON 810 (see review in BPPD 2010a). Both the 1507 x MON 810 pyramid and the single toxin 1507 and MON 810 PIPs are cons idered “high dose” for ECB and likely

SWCB.

For the most part, BPPD agrees with the parameter estimates chosen as the benchmarks for the simulations. Survival of susceptib le genotypes (set at 0.001) on individual Bt toxins was based on high dose expression of both Cry1Ab and Cry1F, which has been demonstrated experimentally for ECB (and pr esumed for SWCB). Resistant individuals were conservatively assumed to have full (100%) survival and no fitness costs on Bt, although this may not hold true for actual fiel d-resistant insects. The initial resistance allele frequency (a sensitive parameter in this model) was set to 0.001 for Cry1Ab, a typical value used for this toxin and ECB in IRM modeling. On the other hand, Pioneer selected a higher value (0.01) for Cry1F and EC B. This was a rational approach in light of some of the annual ECB monitoring resu lts that have been observed since the commercialization of Cry1F corn (see discus sion in BPPD 2010b). Initial resistance allele frequency was clearly a sensitive parameter in the model -- the higher value for Cry1F resulted in substantially less durabi lity relative to the Cry1Ab simulations (33 years for Cry1Ab compared to 11 years for Cry1F).

Cross resistance assumptions for Cry1F were based on Pioneer’s work with resistant colonies (described in the pr evious section of this review ). In those studies, Cry1Fresistant ECB were unable to survive on Cr y1Ab-expressing corn. Considering these results along with the ligand blotting and structural compar isons suggesting independent modes of toxicity, Pioneer assumed no cro ss resistance (i.e., 0% survival of Cry1F resistant ECB on Cry1Ab corn) as the benchm ark default. BPPD believes this could

somewhat underestimate the cross resistance potential given 1) the presence of some shared binding sites in ECB and SWCB in th e ligand blotting experi ments, and 2) the- 19 - susceptibility data (LC 50 ) for Cry1F-resistant ECB on Cry1A b diet (resistance ratio = 10.5). Still, low cross resist ance potential is exp ected for Cry1F and it is unclear (and perhaps unlikely) whether the two factors abov e would have any field relevance. Pioneer addressed this uncertainty by assuming a low level of Cry1F to Cry1Ab cross resistance (0.1) in the worst case scenarios and sens itivity analyses. Although durability was

reduced when levels of cross resistance were increased, the pyramid continued to remain durable relative to the single trait PIPs. A dditional model runs could be conducted to test a higher (hypothetical) level of Cry1F to Cry1Ab cross resistance; however, BPPD can find no empirical basis for doing so.

A greater level of cross resistance was assu med for Cry1Ab (to Cry1F) cross resistance in the benchmark parameters (0.2 for ECB and 0.1 for SWCB). For the worst case and sensitivity scenarios, survival of Cry1Ab-re sistant insects on Cry1F was modeled as high as 0.5 (ECB) and 0.3 (SWCB). As seen in the sensitivity analyses, greater cross resistance negatively affected durability. Howe ver, the parameter was not as sensitive as others; an increase in Cry1A b to Cry1F cross resistance from 0.3 to 0.5 reduced the durability of the pyramid by only 3 years.

Non-compliance was not specifically incl uded as a variable in the comparative

simulations to evaluate 1507 x MON 810 durab ility. In general, non-compliance can be expected to reduce the overall effectiveness of a refuge-based st rategy by increasing the selection pressure for resistance. In a co mparative model, non-compliance could have a differential impact on durability if compliance rates vary for separate refuge options. While there are no data yet to show if co mpliance levels differ between a 20% and 5% refuge, it is reasonable to a ssume that smaller refuges are likely to have greater compliance due to economic and logistical c onsiderations. To e xplore the effects on durability, Pioneer conducted a separate modeling analysis of compliance for 1507 x MON 810 and the single trait PIPs. Complian ce levels were varied from 60 to 100% for ECB and SWCB with the model benchmark parame ters (described in Table 5). As could be expected, increased non-compliance reduced refuge effectiveness (durability) for all PIP types. Nevertheless, for both ECB a nd SWCB the durability of the pyramid (5% refuge) with the lowest rate of non-complian ce (60%) was still consid erably greater than either of the single trait PIPs ( 20% refuge) with 100% compliance.

Pioneer did not include corn earworm (CEW ) in the model analyses reasoning that resistance is unlikely due to: 1) less promin ence in the Corn Belt; 2) no overwintering in the Corn Belt; and 3) highly polyphagous hos t utilization likely provides some natural refuge. BPPD agrees that the resistance poten tial is lower for CEW b ecause of the stated reasons but recognizes that th e insect presents unique resi stance challenges due to lower sensitivity to Bt toxins and an annual cycle th at potentially includes both Bt corn and Bt

cotton. BPPD also notes that Pioneer has a ddressed resistance issues with CEW in a similarly structured model to support an amendment for reduced refuge in cottongrowing areas (reviewed separately).

3) Trait Durability Plan Supporting Reduced Block/Strip Lepidopteran Refuge for 1507 x MON810 Maize in the Corn Belt (MRID# 480056-03)- 20 - Pioneer has proposed a reduced refuge in th e Corn Belt for 1507 x MON 810 corn of 5% (lowered from the 20% refuge requirement im posed for initial product registration). The proposal is based on the following considerations:

i. Dose of 1057 and MON 810. Both of the single tr ait constituent products of 1507 x MON 810 are considered high dose for the two major target pests, ECB and SWCB. Data submitted for the registration of 1507 x MON 810 indicated that the pyramided product also expr esses high dose for ECB and SWCB (reviewed in BPPD 2010).

ii. Independent Modes of Action for Cry1Ab and Cry1F. Previously conducted research and newly submitted studie s (MRID# 480056-01) demonstrate that Cry1Ab and Cry1F operate by independent pa thways in the target pests. This work involved a combination of appro aches to assessing cross resistance including structural comparisons, midgut (receptor) binding assays, pore formation analysis, and tests with Cry1F-re sistant colonies. With discrete modes of action, Cry1Ab and Cry1F produce redunda nt killing in a pyramid, a key to delaying resistance to both toxi ns (as described by Roush 1998).

iii. Improved Efficacy of 1507 x MON 810. Previously submitted (MRID# 476778-03) and new data (Appendix 1, MR ID# 480056-03) show that the 1507 x MON 810 pyramid has comparable efficacy to the single trait PIPs for ECB, SWCB, and FAW. Both the single toxin and combined trait PIPs essentially prevented any measurable feeding damage from the pests. Additionally, ear injury damage from CEW and number of larvae per ear were significantly less among 1507 x MON 810 plants compared to 1507 plants (both PIPs also contained 59122 for corn rootworm).

iv. Effect of Unstructured Refuge on Reducing Selection Pressure. Though not included in the durability analysis, unstru ctured (natural) refuge may serve to reduce selection pressure and dilute re sistance alleles in ECB and SWCB. Natural refuge consists of alternate sources of susceptible insects such as weeds, wild hosts, and other cultivated crops. ECB is polyphagous and is known to feed on a range of wild hosts and crops in cluding strawberry, wheat, sweet corn, popcorn, soybean, snap bean, cotton, smartw eed, ragweed, and cocklebur. In the Corn Belt, ECB infest oats, sorghum, m illet, and potato. Further, a sizable portion of the maize crop in the Corn Belt is planted to non-Bt varieties. SWCB has a smaller host range than ECB that includes corn, sorghum, millet, sugarcane, and Johnson grass. Information is lim ited on the value of these hosts though one study suggests there is less production of SWCB on alternate pl ants relative to

corn.

v. Modeling Predictions for the Durability of 1507 x MON 810 corn. Pioneer’s deterministic model simulations (M RID# 480056-02) showed that 1507 x MON 810 corn with a 5% refuge is much more durable than either single gene 1507 or- 21 - MON 810 with a 20 refuge. The model inco rporated many of the parameters used in Roush’s (1998) model of a pyramided PIP system and also included variables for survival rates (resistant and susceptible), initial resistance allele frequency, dominance, cross resistance, and ref uge size. Durability for 1507 x MON 810 was predicted to be 136 years for ECB and 176 years for SWCB using benchmark parameters. Single trait PIPs were si gnificantly less durable with 33 (MON 810) and 11 (1507) years to resistance for ECB and 22 years for SWCB (both MON 810 and 1507).

vi. Conservative Trait Durability Plan. Pioneer believes that a number of factors make the proposed 5% durability plan for 1507 x MON 810 conservative. Parameters in the modeling included wors t case assumptions of no fitness costs for resistance (most selected colonies show fitness effects) with resistant individuals having no mortality on Bt plan ts (though incomplete resistance would reduce the number of survivors and delay field resistance). Further, Pioneer’s plan assumes complete (100%) adoption of 1507 x MON 810; in actuality, there will be a mosaic of multiple toxins and pyramids, which should serve to lower selection pressure on any one toxin.

Pioneer’s (amended) trait durability plan fo r 1507 x MON 810 corn is intended to cover ECB and SWCB refuge in the Corn Belt. (A separate proposal to reduce refuge in cotton-growing regions is reviewed later in this memorandum). As discussed in the modeling section, CEW was not modeled because of less prominence and low overwintering in the Corn Belt and highly polyphagous hos t utilization.

Other aspects of IRM, incl uding resistance monitoring, remedial action, and compliance monitoring, will follow the plans that have b een established and implemented for Cry1Ab and Cry1F. Grower education will be enhan ced to accommodate a 5% refuge (and avoid grower confusion) through training for deal ers and a “communication rollout plan” for 1507 x MON 810 customers. The overall education plan will follow the existing

framework for Pioneer’s He rculex Bt corn products.

BPPD Review - Trait Durability Plan (5% Refuge)

BPPD recognizes that resistan ce management models and othe r research have shown that a pyramided approach to Bt toxin deploymen t can increase the durability of each toxin relative to mosaics of single toxin PI Ps (Roush 1998, Caprio 1998, Zhao et al. 2003, Gould et al. 2006, Onstad and Meinke 2010). Improved durability in the pyramid is rooted in the “redundant killing” provided by the simultaneous use of multiple toxins against the target pest(s). If each toxin has an independent mode of action with high toxicity against the target, it is extremely diff icult for insects that develop resistance to

one toxin to become resistant to the second. Thus, survival of all genotypes is extremely low which increases the durability of the individual toxins in the pyramid.

Pioneer has submitted data on dose (previously reviewed for initial registration), cross resistance, and simulation modeling to de monstrate that the 1507 x MON 810 pyramid- 22 - has superior durability relative to the singl e trait constituent PIPs. Data for Cry1Ab and Cry1F have been developed using a variet y of approaches (MRID# 480056-01 and other published literature) that have shown that Cr y1Ab and Cry1F have low potential for cross

resistance in ECB and likel y SWCB and CEW. Both toxi ns are known to be highly efficacious in 1507 x MON 810, with high dos e expression for ECB and likely SWCB (BPPD 2010a).

Simulation modeling has shown that even w ith a smaller (5%) refuge, a pyramid of Cry1Ab and Cry1F is substantially more durable than either toxin alone with the standard 20% refuge. For ECB, 1507 x MON 810 was more than four times as durable as MON 810 (136 years vs. 33 years) and 12 times as durable as 1507 (136 years vs. 11 years) with the benchmark model parameters. For SWCB (with benchmark parameters), durability of the pyramid was seven times as durable as either 1507 or MON 810 alone (173 years vs. 22 years). Since most IRM mode ls involve uncertainty with the biological parameter inputs, “worst case” simulations are often performed with extremely conservative assumptions. Pioneer’s wors t case modeling showed that 1507 x MON 810 with a reduced refuge with still more durable than either of the singl e toxin PIPs for each target pest (Table 6).

Pioneer presented a number of arguments as to why their proposed trait durability plan (with a 5% refuge) for 1507 x MON 810 is scientif ically justified yet still a conservative IRM approach. In addition to high dose for th e target pests, low cross resistance potential for the toxins, and simulation modeling, the company cited the increased efficacy of the pyramid, possible natural refuge for ECB and SWCB in the Corn Belt, and the presence of a toxin mosaic in the landscape from multip le PIP registrations (Pioneer’s analysis assumed 100% 1507 x MON 810). BPPD conclude s that these factor s are reasonable and provide support for a reduced refuge, with th e exception of the natural refuge claim. Although ECB and SWCB are known to be polyphagous, it is unknown whether any

alternate hosts in the Corn Belt would f unction as actual refuge (i.e. a source of susceptible insects). An investigation of a lternate hosts would be needed to determine production of ECB/SWCB (relative to corn), availability of non-corn hosts in the landscape (including different geographies within the Corn Belt), and a temporal analysis

of ECB/SCWB utilization of these hosts relative to corn. Such an assessment was performed for Bt cotton that included extensive sampling of the target pests and a novel bioassay to determine host origin (see Orth et al. 2007, Jackson et al. 2008); a similar approach would likely be needed for ECB and SWCB.

Should 1507 x MON 810 be registered with a 5% refuge requirements for lepidopteran pests, the product will have different re fuge requirements than many of the other registered Bt corn products that have a 20% refuge. Therefore, additional aspects of the IRM program for 1507 x MON 810 including compliance and grow er education will need to be modified (or developed) due to accommodate the different refuge size. Pioneer has proposed additional training for seed dealers and a communication plan for customers. BPPD supports these measures , but also recommends increased emphasis on information supplied to growers, who must ma nage the refuge requirements. BPPD notes that the refuge reduction has been proposed only for lepidopteran-protected 1507 x MON- 23 - 810 corn; however, if stacked corn rootworm products are eventu ally included (that require a separate 20% refuge) an additional burden and source of confusion may result for growers. It is recommended that Pioneer submit revised copies of their grower use guide to EPA to verify that the proper ref uge instructions are included. In terms of compliance monitoring and the establishe d Compliance Assurance Program (CAP), Pioneer may need to revise definitions of “s ignificant” deviations with a 5% refuge.

BPPD recommends that Pioneer remove the “o ther” deviations categ ory and define any amount of non-compliance with the reduced refuge as “significant.”

BPPD agrees that the existing programs fo r resistance monitoring and remedial action that were established for MON 810 (Cry1Ab) and Herculex (Cry1F) should be applicable

to 1507 x MON 810 corn.

References

BPPD, 2010a. Review of Insect Resistance Management (IRM) considerations for Bt corn events 1507 x MON 810, 59122 x MON 810, 1507 x 59122 x MON 810. Memorandum from A. Reynolds to A. Sibold, dated January 7, 2010.

BPPD, 2010b. Biopesticide Registration Action Document: Cry1Ab and Cry1F Bacillus thuringiensis (Bt) Corn Plant-Incorporated Prot ectants. Updated September, 2010.

Caprio, M., 1997. Corn-cotton ecosystem model for resistance evolution in Helicoverpa

zea to Bt-endotoxins expressed in plants. Unpublished study submitted to EPA (part of

Novartis’ 1997 IRM research report). MRID # 444754-01.

Caprio, M., 1998. Evaluating resistance management strategies for multiple toxins in the presence of external refuges. J. Econ. Entomol. 91: 1021-1031.

Crespo, A.L.B., T.A. Spencer, A.P. Alves, R.L. Hellmich, E.E. Blankenship, L.C. Magalhaes, and B.D. Siegfried, 2009. On-plant survival and inheritance of resistance to Cry1Ab toxin from Bacillus thuringiensis in a field-derived strain of European corn borer, Ostrinia nubilalis

. Pest Man. Sci. 65: 1071-1081.

Gould, F., M. B. Cohen, J. S. Bentur, G. C. Kennedy, and J. Van Duyn, 2006. Impact of small fitness costs on pest adaptation to crop varieties with multiple toxins: a heuristic

model. J. Econ. Entomol. 99: 2091-2099.

Granero, F., V. Ballester, and J. Ferre, 1996. Bacillus thuringiensis crystal proteins CRY1Ab and CRY1Fa share a high affinity binding site in Plutella xylostella (L.). Biochem. Biophys. Res. Comm. 224: 779-783.

Hua, G., L. Masson, J.L. Jurat-Fuentes, G. Schwab, and M.J. Adang, 2001. Binding Analyses of Bacillus thuringiensis Cry {delta}-Endotoxins Using Brush Border

Membrane Vesicles of Ostrinia nubilalis

. Appl. Environ. Microbiol. 67: 872-879.- 24 - Jackson, R. E., J. R. Bradley, J. W. Van Duyn, B. R. Leonard, R. Luttrell, J. Ruberson, J. J. Adamczyk, J. Gore, D. Hardee, R. Voth, S. Sivasupramaniam, J. W. Mullins and G. Head, 2008. Regional assessment of Helicoverpa zea (Lepidoptera: Noctuidae) populations on cotton and non-cotton crop hosts. Entomol. Exp. Appl. 126: 89-106.

Onstad, D. and L. Meinke, 2010. Modeling Evolution of Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) to Transgenic Corn with Two Insecticidal Traits. J. Econ

Entomol. 103: 849-860.

Orth, R., Head, G., and M. Mierkowski, 2007. Determining Larval Host Use by a Polyphagous Lepidopteran through Analysis of Adult Moths for Plant Secondary

Metabolites. J. Chem. Ecol. 33: 1131-1148.

Pereira E.J., B.A. Lang, N.P. Storer, and B.D. Siegfried, 2008. Selection for Cry1F resistance in the European corn borer a nd cross-resistance to other Cry toxins. Entomol. Expt. Appl. 126: 115-121.

Pereira E.J., H.A. Siqueira, M. Zhuang, N.P. Storer, B.D. Siegfried, 2010. Measurements of Cry1F binding and activity of luminal gut pr oteases in susceptible and Cry1F resistant

Ostrinia nubilalis larvae (Lepidoptera: Crambidae). J. Invertebr. Pathol. 103: 1-7.

Roush, R.T., 1998. Two toxin strategies for manage ment of insecticidal transgenic crops: will pyramiding succeed where pesticide mixtures have not? Phil. Trans. R. Soc. Lond.

353: 1777-1786.

Siqueira, H.A.A., D. Moellenbeck, T. Spencer, and B.D. Siegfried, 2004. Crossresistance of CrylAb-selected Ostrinia nubilalis (Lepidoptera: Crambidae) to Bacillus thuringiensis d-endotoxins. J. Econ. Entomol. 97: 1049-1057.

Zhao, J., J. Cao, Y. Li, H. Collins, R. Rous h, E. Earle, and A. Shelton, 2003. Transgenic plants expressing two Bacillus thuringiensis toxins delay insect resistance evolution. Nature Biotechnology. 21: 1493-1497.