October 20, 2010 

MEMORANDUM

SUBJECT: Review of Insect Resistance Management (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

BPPD1

 has been asked to review Insect Resistance 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 Belt. 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 No. 29964-7) from 20% to 5% in the Corn 

 

1

 The use of BPPD in this review refers to the BPPD 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 potential for Cry1Ab and Cry1F, and 

simulation modeling for two major target pests, 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 toxin 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 resistance potential in ECB and likely SWCB and 

corn earworm (CEW). Pioneer employed a range of approaches to investigate Cry1F - 

Cry1Ab cross resistance including structural comparisons, midgut (receptor) binding 

assays, pore formation analysis, and tests with 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 resistance. On the other hand, experiments with 

Cry1F-resistant ECB showed no survival on Cry1Ab. The potential for low level cross 

resistance between Cry1F and Cry1Ab was addressed by exploring a range of values 

using a sensitivity analysis with the simulation model. 

3) Pioneer’s simulation model showed that even with a smaller (5%) refuge, the 1507 x 

MON 810 pyramid is substantially more durable than either 1507 (Cry1F) or MON 810 

(Cry1Ab) 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) 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 the Corn Belt refuge proposal. BPPD agrees 

with Pioneer that resistance is unlikely for this 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 compliance will need to be modified (or 

developed) to accommodate the different refuge size. BPPD supports the educational 

measures proposed by Pioneer, but also recommends increased emphasis on information 

supplied to growers, who must manage the refuge requirements. It is further 

recommended that Pioneer submit revised copies 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 requirements for resistance monitoring, remedial 

action, and annual reporting be maintained 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 products containing the Cry1F (event 1507) 

and Cry1Ab (event MON 810) toxins, which were derived by conventional breeding 

from Pioneer’s Herculex Cry1F product (EPA 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 and 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 lepidopteran 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 Management (IRM) considerations for 1507 x MON 

810 (see BPPD 2010a) including data on dose (field efficacy and protein expression 

studies). Previous IRM assessments for 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% refuge 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 refuges to 5% (from 20%) in the Corn Belt for 

1507 x MON 810 corn only (29964-7). The other 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 the 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 Supporting 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 References 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 reviewed separately and is not addressed in 

this memorandum. 

Proposal for 5% Refuge in the Corn Belt

1) Mode of Action and Cross Resistance 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 toxins act independently (without 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 are involved in midgut 

receptor recognition and pore formation. Results from the alignment analysis showed 

that Cry1Ab and Cry1F have <74% amino acid protoxin sequence homology. Domain 

sequence similarities between the two toxins were 68% (DI), 46% (DII), and 62% (DIII). 

Pioneer concluded that the low relative sequence similarity supports the prediction that 

the two proteins utilize different midgut receptor sites. 

Pioneer also provided a structural comparison using three dimensional models of Cry1Ab 

and Cry1F (developed from the structural coordinates 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 were 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 binding 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 Cry1Ab 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 binding interactions. Binding studies were 

conducted using Brush Border Membrane Vesicle (BBMV) protein obtained from 

dissected ECB, CEW, or SWCB midguts. Cry1Ab toxin (prepared using the 

methodology described in Hua et al. 2001) was labeled with Na125I for detection by 

radioactivity counters. Cry1F toxin used in the competitive binding testing (mature, 

trypsin activated; as with Cry1Ab) was marked using a fluorescent labeling technique 

(Alexa-Fluor) for detection by SDS-PAGE and an imaging device (Typhoon Trio 

Variable Mode Imager). Ligand blots were performed using BBMV proteins separated 

by SDS-PAGE and fixed on nitrocellulose membranes. Each membrane was incubated 

with either Cry1Ab or Cry1F toxin followed by incubation with anti-Cry1Ac or antiCry1F antibodies. Secondary (goat anti-rabbit) antibodies with fluorescent labeling were 

used for detection of the bound protein. Western blots were also conducted using similar 

procedures to detect aminopeptidase-N (APN) and alkaline phosphatase (ALP) proteins 

that could function as toxin receptors. The 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 Appendix 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) was observed at the higher concentrations of Cry1F. 

Conversely, homologous competition (Cry1Ab/Cry1Ab) showed approximately 90% less 

125I-Cry1Ab binding at the maximum concentrations. A different pattern of results were 

observed with the reciprocal Cry1Ab competition 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 during the Cry1Ab-Cry1F exposure (suggesting shared and 

unshared binding sites). However, for Cry1F-resistant ECB, the presence of Cry1F did 

not significantly inhibit Cry1Ab binding indicating that the shared 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 binding proteins within ECB, SWCB, and CEW 

BBMVs for both Cry1Ab and Cry1F. A summary 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 

SWCB2

25 (ALP) 

50 

60 (ALP) 

110 

120 

180 (APN) 

210 

>250 

110 

120 

130 (APN) 

180 (APN) 

>250 

CEW3

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 inhibition in the presence of Cry1Ab, though when Cry1F was 

introduced as a competitor to Cry1Ab binding sites no competition was observed. One 
- 7 -

possibility could be that Cry1F has fewer 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 binding 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 binding sites despite the presence of some 

shared binding proteins. Since multiple toxin pathways (receptors) exists, there is the 

potential for “redundant killing” 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, SWCB, and CEW: one that is 

recognized by Cry1F and a second that is not recognized by Cry1F. Pioneer indicated 

that the findings in their research supported similar conclusions reached by Hua et al. 

(2001). 

c) Pore formation analysis (toxin activity) 

Toxin activity by pore formation was assessed by using short circuit current 

measurements of dissected insect midguts. The assay is designed so that midgut tissue is 

mounted in a two-chambered arena (Ussing-style 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 placed on either side of the midgut tissue to 

measure current (i.e., the movement of potassium 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 response (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 midguts (4.4 min Cry1F-resistant vs. 2.1 min 

susceptible). Finally, Cry1F-resistant midguts 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 demonstrated that there is no 

antagonism between the two toxins. 

d) Cry1F-resistant insect experiments

In addition to the physiological and structural assessments of Cry1Ab and Cry1F cross 

resistance, Pioneer conducted bioassays with Bt-resistant ECB. Two separate projects 

were conducted using several ECB colonies 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 from Iowa and reared for seven generations) 

and Cry1F-resistant ECB colony were included in the testing. The resistant colony was 

originally collected from Hamilton County, Iowa in 2004 (held for 33 subsequent 

generations in culture) and has 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 advanced past the first instar were considered dead) and any 

surviving larvae were weighed. 

As shown in Table 2 below, both susceptible and Cry1F-resistant larvae exposed to 

isoline (non-Bt corn) showed relatively low mortality (21% susceptible and 14% 

resistant). On Cry1F (1507) leaf discs, the 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 resistant vs. 0.350 mg 

susceptible) and also comparable to the weights from the isoline corn trials. This 

indicates that the susceptible larvae surviving Cry1F exposure likely experienced little 

fitness costs. On the other hand, complete mortality was observed for all ECB larvae 

(susceptible and resistant colonies) that were exposed to the Cry1Ab (MON 810) and 

pyramid (Cry1Ab x Cry1F) treatments. Based on these results, the study authors 

concluded that a pyramid of Cry1F and Cry1Ab provides redundant killing of ECB even 

for Cry1F-resistant larvae. 

Table 2. Susceptibility of Cry1F-Resistant and Reference ECB Colonies to Cry1Ab and 

Cry1F in Single and Pyramid Treatments (taken 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 further genetically characterize a Cry1Fresistant strain in terms of inheritance of resistance, sex-linkage, dominance, resistance 

loci, and cross resistance with Cry1Ab. Testing was conducted using diet bioassays and 

susceptible and Cry1F-resistant ECB colonies. Resistant ECB (originally obtained from 

collections in South Dakota, Minnesota, and Nebraska) were selected for at least 15 

generations on Cry1F in the laboratory (reported to be the highest Bt resistance level 

achieved for ECB). Susceptible ECB (to both Cry1F and Cry1Ab) were collected from 

Iowa and maintained for at least 10 generations in culture. Reciprocal and backcrosses 

were also created from matings of the susceptible and Cry1F-resistant colonies. 

Bioassays were conducted using diet overlays of varying amounts (serial dilutions) of 

Cry1F or Cry1Ab toxin (untreated controls were also tested). Neonate ECB larvae were 

exposed for a seven day period after which mortality was assessed. Dead larvae included 

individuals that failed to advance past the first instar or weighed less than 0.1 mg. 

Reciprocal crosses between susceptible and resistant ECB were used to investigate sex 

linkage and dominance while backcrosses (of the reciprocal crosses with the Cry1Fresistance colony) were used to determine the number of resistance loci. 

Results from the experiment showed that the Cry1F-resistant ECB colony was highly 

tolerant of Cry1F (LC50 >24,600 ng/cm2

) compared to the susceptible colony (LC50 = 

0.43 ng/cm2) giving a resistance ratio of >57,500 (Table 3). Reciprocal crosses showed 

somewhat greater tolerance of Cry1F than the susceptible colonies (LC50 = 1.34 ng/cm2 

for R♂x S♀, 1.25 ng/cm2 for S♂x R♀, and 1.38 ng/cm2 for the pooled cross). Given the 

similar responses among the F1 reciprocal crosses, inheritance appears to be autosomal 

and not sex-linked. For the susceptible (SS), resistant (RR), and reciprocal crosses (F1

pooled, RS) dominance was estimated (as a function of the survival of each genotype at 

each concentration tested). At the higher bioassay concentrations (e.g, 8.0 and 40.0 

ng/cm2

), dominance was low indicating recessive resistance. However, at lower 

concentrations (0.32 and 1.6 ng/cm2), higher dominance values (close to 1.0) suggest 

almost complete dominance. A Chi Square test 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 concentrations (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 crosses (Table 3). The susceptible colony 
- 10 -

remained highly sensitive to Cry1Ab but the Cry1F-resistant colony showed some 

tolerance to Cry1Ab (resistance ratio = 10.5). However, the reciprocal crosses were 

almost as sensitive to Cry1Ab as the susceptible colony (resistance ratios < 1.21). 

Additional testing with leaf discs showed that the Cry1F-resistant colony (tolerant of 

Cry1F leaf discs) was completely controlled by leaf discs containing Cry1Ab or a 

pyramid of Cry1F and Cry1Ab. 

Table 3. Responses of Susceptible and Cry1F-Resistant ECB Colonies and Reciprocal 

Crosses to Cry1F and Cry1Ab Diet Bioassays (taken from data provided in Tables 1 and 

4 of Appendix 4 in MRID# 480056-01) 

ECB Colony 

Cry1F Cry1Ab 

LC50 (ng/cm2) Resistance 

Ratio LC50 (ng/cm2

) Resistance 

Ratio 

Susceptible (S) 0.43 -- 0.051 -- 

Cry1F Resistant (R) > 24,600 > 57,500 0.534 10.5 

Reciprocal Cross - 

R♂ x S♀ (F1) 1.34 3.14 0.061 1.19 

Reciprocal Cross - 

S♂ x R♀ (F1) 1.25 2.94 0.054 1.07 

Reciprocal Cross - 

pooled (F1) 1.38 3.24 0.062 1.21 

Based on the data, the study author concluded that Cry1F (1507) and Cry1Ab (MON 

810) combined in a pyramid can extend trait durability. Low level cross resistance 

observed in the Cry1Ab diet bioassays with the Cry1F-resistant strain are not likely to be 

significant enough to allow field survival (as evidenced 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 submitted data provide a scientific weight of 

evidence to support the conclusion that Cry1Ab 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 cross resistance criterion for simultaneous 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 binding characteristics, toxin activity (pore 

formation) analysis, and studies with Cry1F-resistant insect colonies. These data largely 

show that Cry1Ab and Cry1F elicit toxic responses through separate pathways and 

support the findings of previously 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 presence of several shared binding proteins 

(25 and 150 kDa) in ECB although numerous other unshared (unique) binding proteins 

were also identified. On the other hand, the presence of Cry1F in competitive binding 

assays did not inhibit Cry1Ab binding with BBMV proteins. A second test also showed 

that for a Cry1F-resistant ECB, Cry1F did not significantly affect Cry1Ab binding 

indicating that an unshared Cry1Ab binding site still remained viable. 

The binding work conducted by Pioneer largely 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 kDas 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 concentrations) 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 high affinity site (unrelated to Cry1Ab) 

responsible for toxicity in ECB. Separate research conducted by Pereira et al. (2010) 

found that Cry1F recognizes binding sites 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 resistance 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 for the Cry1F/Cry1Ab binding results including 

the possibility that Cry1F has fewer binding receptors than Cry1Ab (though Cry1F has 

shown to be efficacious in the field) or that Cry1F has an additional unique binding site 

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

evidence of low level cross resistance through the presence of shared binding sites in 

ECB BBMV. However, other submitted and published data largely corroborate the view 

that both toxins likely recognize unique sites that cause most of toxicity in ECB. 

Further evidence supporting lack of Cry1F/Cry1Ab cross resistance was provided in 

studies conducted with Cry1F-resistant ECB. Pore formation analysis with Cry1Fresistant and susceptible ECB showed that channels 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 indicates that Cry1Ab should still be 

effective against Cry1F-resistant ECB. 
- 12 -

Susceptibility studies (leaf disc and diet bioassays) with Cry1F-resistant ECB also 

showed that Cry1Ab still remained effective against these insects. Cry1Ab-expressing 

leaf discs (as a single 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 unselected control colony (resistance ratio = 

10.5). This result could be due to the presence 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 level cross resistance is unlikely to be field 

relevant. The findings of Pioneer were largely 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 buttressed 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 whether such colonies were (or are) available 

to Pioneer for testing. Pioneer reasoned that such testing should not be undertaken 

because Cry1Ab resistance (polygenic) is different than Cry1F resistance (monogenic) 

and Cry1Ab selection results in moderate resistance (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 toxin 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 levels 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 pathways 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 (Table 1). Unlike the ECB work, separate 

studies with these insects were 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 resistance potential in CEW and 

SWCB with little or no field relevance, similar to ECB. 

2) Predictive Model Supporting Reduced Block/Strip Lepidopteran Refuge for 1507 x 

MON810 (MRID# 480056-02)

To evaluate the durability of a reduced (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. Pioneer employed an approach similar to that 

taken by Roush (1998) in terms of parameter assumptions. A comparison of the two 

modeling approaches was provided by Pioneer 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 (table reproduced verbatim 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-transgenic hosts survive equally well as 

those laid by resistant homozygous 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 reviewed literature). For 

many of the potential genotypes, SWCB was assumed to respond similarly to ECB (in 

other cases, parameters were varied for SWCB). Homozygous resistant individuals were 

assumed to have 100% survival on their corresponding single toxin isolines. Both 

homozygous susceptible and heterozygous insects were presumed to have 0% survival on 

Bt corn, with the exception of Cry1F resistant heterozygotes whom were assumed to have 

0-5% survival on Cry1F corn. Cross resistance survival of Cry1F-resistant ECB on 

Cry1Ab was set to zero based on work with resistant colonies (Pereira et al. 2008; see 

discussion in the previous section). For Cry1Ab resistance, survival on Cry1F was set at 

0.2 for ECB and 0.1 for SWCB; conservative levels of cross resistance that Pioneer 

believes exceed those observed in Siqueira et al. (2004). Single toxin 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 assumed 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 and 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 scenarios with “best,” “worst,” and “benchmark” 

scenarios. Benchmark simulations were based upon empirical data and literature 

(described above and in the previous section) and were considered 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, were conducted by varying one 

parameter while holding the others steady. Separate analyses were conducted for the 

cross resistance, dominance, susceptible survival 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 frequencies 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 parameters 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 benchmark 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 incorporating best case parameters resulted 

in greater relative durability particularly for the Cry1F x Cry1Ab pyramid -- no resistance 

evolved among either ECB or SWCB within 400 generations (200 years). 

Comparatively, resistance developed to Cry1Ab 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 (Cry1Ab) 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 durability. The company believes the modeling 

analysis is conservative given 1) polyphagous feeding by the target pests, particularly 

ECB (not addressed in the model), 2) the assumption 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 ECB parameter estimates of cross resistance, 

survival of susceptible insects, initial resistance allele frequency, and dominance (output 

summarized in Table 7). A range of high, medium (i.e., benchmark), and low estimates 

were incorporated into simulations to estimate 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 years durability with ECB. The model was 

sensitive to all four parameters as measured by the relative ranges of durability between 

the high and low estimates. Durability was greatly 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 estimates for initial 

resistance allele frequency and cross resistance. The model was generally less sensitive 

to the lower values used for survival of susceptible insects and dominance (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 parameter assumptions chosen, a pyramid of 

1507 (Cry1F) and MON 810 (Cry1Ab) has superior durability to single trait PIPs 

expressing Cry1Ab and Cry1F. In all scenarios 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 greater than the single trait products and a 

20% refuge (see Table 6). For ECB, the pyramid 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 frequency assumptions for 

Cry1Ab and Cry1F were probably responsible for the contrast in durability between the 

single toxin events. With SWCB, the benchmark simulations showed that 1507 x MON 

810 was more than 7 times as durable as either 1507 or MON 810 alone (173 years vs. 22 

years). In the best case scenarios, resistance 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 regarding the model input. With many IRM 

simulations, parameters such as initial resistance 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 employing highly conservative assumptions, 
- 18 -

particularly for Cry1Ab-Cry1F cross resistance, survival of susceptibles on the Bt toxins, 

and dominance of the resistance alleles. These 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% refuge): for ECB, 9 years compared to 8 

years (MON 810) and 3 years (1507); for SWCB, 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 evolution of resistance to the single toxin PIPs (8 years or less). 

In fact, both Cry1Ab (registered since 1995) and Cry1F (registered since 2001) have been 

in commercial use for much longer than the predicted time to resistance in the worst case 

modeling. 

Output from Pioneer’s simulations largely corroborates other modeling efforts that have 

examined pyramided approaches to resistance 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 evolution of resistance relative to single toxin deployments. 

Lack of cross resistance has been addressed by Pioneer in this submission (refer to the 

previous section); dose was previously assessed for the initial registration 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 considered “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 susceptible 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 presumed 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 field-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 ECB. This was a rational approach in light 

of some of the annual ECB monitoring results that have been observed since the 

commercialization of Cry1F corn (see discussion in BPPD 2010b). Initial resistance 

allele frequency was clearly a sensitive parameter in the model -- the higher value for 

Cry1F resulted in substantially less durability 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 previous section of this review). In those studies, Cry1Fresistant ECB were unable to survive on Cry1Ab-expressing corn. Considering these 

results along with the ligand blotting and structural comparisons suggesting independent 

modes of toxicity, Pioneer assumed no cross resistance (i.e., 0% survival of Cry1F 

resistant ECB on Cry1Ab corn) as the benchmark 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 the ligand blotting experiments, and 2) the 
- 19 -

susceptibility data (LC50) for Cry1F-resistant ECB on Cry1Ab diet (resistance ratio = 

10.5). Still, low cross resistance potential is expected for Cry1F and it is unclear (and 

perhaps unlikely) whether the two factors above 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 sensitivity analyses. Although durability was 

reduced when levels of cross resistance were increased, the pyramid continued to remain 

durable relative to the single trait PIPs. Additional 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 assumed 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-resistant 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. However, the parameter was not as sensitive as 

others; an increase in Cry1Ab 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 included as a variable in the comparative 

simulations to evaluate 1507 x MON 810 durability. In general, non-compliance can be 

expected to reduce the overall effectiveness of a refuge-based strategy by increasing the 

selection pressure for resistance. In a comparative 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 compliance levels differ between a 20% and 5% 

refuge, it is reasonable to assume that smaller refuges are likely to have greater 

compliance due to economic and logistical considerations. To explore the effects on 

durability, Pioneer conducted a separate modeling analysis of compliance for 1507 x 

MON 810 and the single trait PIPs. Compliance levels were varied from 60 to 100% for 

ECB and SWCB with the model benchmark parameters (described in Table 5). As could 

be expected, increased non-compliance reduced refuge effectiveness (durability) for all 

PIP types. Nevertheless, for both ECB and SWCB the durability of the pyramid (5% 

refuge) with the lowest rate of non-compliance (60%) was still considerably 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 prominence in the Corn Belt; 2) no overwintering in 

the Corn Belt; and 3) highly polyphagous host utilization likely provides some natural 

refuge. BPPD agrees that the resistance potential is lower for CEW because of the stated 

reasons but recognizes that the insect presents unique resistance challenges due to lower 

sensitivity to Bt toxins and an annual cycle that potentially includes both Bt corn and Bt 

cotton. BPPD also notes that Pioneer has addressed 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 the Corn Belt for 1507 x MON 810 corn of 5% 

(lowered from the 20% refuge requirement imposed for initial product registration). The 

proposal is based on the following considerations: 

i. Dose of 1057 and MON 810. Both of the single trait 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 expresses 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 studies (MRID# 480056-01) demonstrate that 

Cry1Ab and Cry1F operate by independent pathways in the target pests. This 

work involved a combination of approaches to assessing cross resistance 

including structural comparisons, midgut (receptor) binding assays, pore 

formation analysis, and tests with Cry1F-resistant colonies. With discrete modes 

of action, Cry1Ab and Cry1F produce redundant killing in a pyramid, a key to 

delaying resistance to both toxins (as described by Roush 1998). 

iii. Improved Efficacy of 1507 x MON 810. Previously submitted (MRID# 

476778-03) and new data (Appendix 1, MRID# 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, unstructured (natural) refuge may serve to 

reduce selection pressure and dilute resistance 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 including strawberry, wheat, sweet corn, 

popcorn, soybean, snap bean, cotton, smartweed, ragweed, and cocklebur. In the 

Corn Belt, ECB infest oats, sorghum, millet, 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 limited on the value of these hosts though one 

study suggests there is less production of SWCB on alternate plants relative to 

corn. 

v. Modeling Predictions for the Durability of 1507 x MON 810 corn. Pioneer’s 

deterministic model simulations (MRID# 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 incorporated 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 refuge 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 significantly 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 worst case assumptions of no fitness costs 

for resistance (most selected colonies show fitness effects) with resistant 

individuals having no mortality on Bt plants (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 for 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 host utilization. 

Other aspects of IRM, including resistance monitoring, remedial action, and compliance 

monitoring, will follow the plans that have been established and implemented for Cry1Ab 

and Cry1F. Grower education will be enhanced to accommodate a 5% refuge (and avoid 

grower confusion) through training for dealers and a “communication rollout plan” for 

1507 x MON 810 customers. The overall education plan will follow the existing 

framework for Pioneer’s Herculex Bt corn products. 

BPPD Review - Trait Durability Plan (5% Refuge)

BPPD recognizes that resistance management models and other research have shown that 

a pyramided approach to Bt toxin deployment can increase the durability of each toxin 

relative to mosaics of single toxin PIPs (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 difficult 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 demonstrate that the 1507 x MON 810 pyramid 
- 22 -

has superior durability relative to the single trait constituent PIPs. Data for Cry1Ab and 

Cry1F have been developed using a variety of approaches (MRID# 480056-01 and other 

published literature) that have shown that Cry1Ab and Cry1F have low potential for cross 

resistance in ECB and likely SWCB and CEW. Both toxins are known to be highly 

efficacious in 1507 x MON 810, with high dose expression for ECB and likely SWCB 

(BPPD 2010a). 

Simulation modeling has shown that even with 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 models involve uncertainty with the biological 

parameter inputs, “worst case” simulations are often performed with extremely 

conservative assumptions. Pioneer’s worst case modeling showed that 1507 x MON 810 

with a reduced refuge with still more durable than either of the single 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 scientifically justified yet still a conservative 

IRM approach. In addition to high dose for the 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 multiple PIP registrations (Pioneer’s analysis 

assumed 100% 1507 x MON 810). BPPD concludes that these factors are reasonable and 

provide support for a reduced refuge, with the 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 function as actual refuge (i.e. a source of 

susceptible insects). An investigation of alternate 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 refuge 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 grower 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 manage 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 eventually 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 refuge instructions are included. In terms of 

compliance monitoring and the established Compliance Assurance Program (CAP), 

Pioneer may need to revise definitions of “significant” deviations with a 5% refuge. 

BPPD recommends that Pioneer remove the “other” deviations category and define any 

amount of non-compliance with the reduced refuge as “significant.” 

BPPD agrees that the existing programs for 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

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BPPD, 2010b. Biopesticide Registration Action Document: Cry1Ab and Cry1F Bacillus 

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

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