CONCLUSIONS AND RECOMMENDATIONS 

BPPD has reviewed Pioneer/DuPont’s application for a 5% Optimum AcreMax seed blend 

(Cry1Ab and Cry1F) and 10% Optimum AcreMax Xtra seed blend (Cry1Ab, Cry1F, and 

Cry34/35) targeting lepidoptera pest of corn (ECB and SWCB) and has concluded that there are 

a number of concerns and uncertainties with the proposed refuge strategies. Because of the 

uncertainties, BPPD is unable to quantify the relative durability of the proposed seed blends 

compared with current structured refuge requirements (5% pyramided toxins or 20% for single 

toxins in deployment). 

Based on simulation modeling, the applicant reported that the durability of a 5% seed blend and 

5% structured refuge strategy for both pests exceeded 200 years when benchmark parameter 

assumptions were used, while they estimated that population extinction could be reached in less 

than 10 years. BPPD concluded that the importance of genetic drift should not be ignored when 

populations are below one individual/hectare and that the Pioneer/DuPont’s durability estimates 

are therefore uncertain. BPPD made recommendations on how to address this uncertainty. Under 

the applicant’s worst-case model assumptions (actual durability estimates were provided in this 

case and given in years to resistance) and Pioneer/DuPont’s model construct, there was a 12.5 

fold loss in durability for a 5% OAM seed blend targeting SWCB compared to a 5% structured 

refuge. The SAP (2011) recommended that IRM strategies should be designed around the pest 

complex at greatest risk of evolving resistance. BPPD concludes that for OAM, the main focus 

may likely have to be on an IRM strategy for SWCB. BPPD also concluded that some parameter 

values needed to be more conservative (i.e., cross resistance, several assumptions for dominance) 

and other important aspects of larval movement and survival were not addressed (i.e., NBI 

hypothesis, epistatic effects). 

An SAP meeting (December 2010), convened to evaluate a similar seed blend product, 

concluded (based on modeling analysis) that a dual gene, high efficacy product with a seed blend 

approach targeting ECB and SWCB was less durable than a block refuge of equivalent non-Bt 

proportion (SAP 2011). At this time, the Agency is unable to confidently quantify the relative 

durability for a seed blend strategy with two toxins compared with the 5% structured refuge 

using the applicant’s and/or SAP’s model construct. All models (whether complex or simple) are 

idealizations and abstractions of reality, have incorporated those mechanisms and assumptions 

that the model designers feel are important, and contain an inherent amount of uncertainties. The 

SAP (2011) therefore recommended that more attention should be paid to relative comparisons 

between various model outputs rather than their absolute values (years of durability). 

Additionally, the applicant did not submit any modeling for a 10% OAM Xtra blend, which is a 

stack of the OAM pyramided toxins and Cry34/35Ab1 (aimed at controlling both lepidoptera and 

corn rootworm pests of corn). In light of the uncertainties with the 5% seed blend proposal 

mentioned throughout this review, such an additional modeling analysis would have allowed the 

Agency to assess the durability of a greater blend percentage. Generally, BPPD would expect a 

10% seed blend to be more conservative and more durable than a respective 5% seed blend with 

two lepidoptera toxins; however, BPPD can not quantify the durability without a comparative 

analysis. 

The Agency previously assessed the risk of a single toxin seed blend strategy for CRW (5% and 

10%), and BPPD conclusions are contained in BPPD 2008, 2010a, and 2010b. This current 

assessment presents only conclusion for the lepidoptera portion of a 5% seed blend strategy. 

- 2 - 
BPPD recommends that Pioneer/DuPont incorporate the following changes as discussed 

throughout the review (and summarized below) into their model, provide a comparative 

modeling analyses, and address some of the remaining biological uncertainties. 

• Report final resistance allele frequency and actual estimates of durability rather than 

‘greater than’ because relative comparisons between various strategies may be 

meaningful even if actual numbers are not.. 

• Artificially inflate fecundity of the pest to 1) avoid extinction, 2) introduce importance of 

genetic drift, and 3) determine relative durability for a seed blend and structured refuge. 

• Incorporate a probability analysis to address uncertainties with various important 

parameter values and to provide the Agency with a risk profile (i.e., 0%, 5%, and 50%) 

for OAM and the modeled IRM strategies as well as a level of significance between 

estimated durability for different strategies. 

• Provide comparative modeling (incorporating BPPD’s recommendations in this review) 

to evaluate: 

- 20% block refuge (single toxin) 

- 5% block refuge (pyramid) 

- 5% blended refuge (pyramid) 

- 10% blended refuge (pyramid) 

• Correct the dominance value for pre-dispersal mortality/survival to be reflective of a lowdose exposure rather than a high-dose exposure. 

• Include non-uniform oviposition parameter for first generation lepidoptera in OAM Xtra 

and second generation adults in OAM seed blends. 

• Include NBI-movement hypothesis (SAP 2011) to address mortality associated with RS 

genotypes that originate and remain during most of their development on non-Bt but 

move as late-instars onto Bt where they have a certain probability of survival. 

• Research on adult movement (related to mating and movement from refuges), larval 

movement, larval feeding (i.e., selective feeding within corn ears or on pollen), survival 

of heterozygote genotypes on OAM (markers may need to be determined for 

heterozygotes), and the potential for epistatic mechanisms of resistance (particularly with 

older instars). 

 

- 3 - 
I. BACKGROUND 

Bt corn event 1507 x MON 810 (EPA Reg. No. 29964-7) and 1507 x 59122 x 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 Cry34/35Ab1 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. The registration was 

granted with a 20% refuge in the Corn Belt and a 50% refuge in cotton-growing areas in the 

southeastern United States. 

Pioneer subsequently proposed an amendment for a reduction in structured refuge for Bt corn 

event 1507 x MON 810 in the Corn Belt from the standard 20% to 5%. After review, EPA 

approved this amendment in October of 2010. 

In this application, Pioneer submitted a proposal to change the IRM strategy from a 5% block 

refuge to a 5% seed blend for 1507 x MON 810 (Optimum AcreMax, OAM) and a 10% seed 

blend for 1507 x 59122 x MON 810 (Optimum AcreMax Xtra, OAM Xtra). Supporting data for 

this request are contained in MRIDs 480707-01, 480707-02, and 481262-01. 

Optimum AcreMax1 Insect Protection (OAM1) is a precursor to the requested Optimum 

AcreMax Xtra registration. In February, 2008, Pioneer Hi-Bred International, Inc. submitted the 

original application to register OAM1, which is a corn seed blend product that contains two 

different kinds of seeds from the already registered products Herculex XTRA and Herculex I. 

OAM1 incorporated the CRW refuge into the seed bag. In addition to switching to a seed mix 

paradigm, Pioneer requested a reduction in refuge for CRW from the standard 20% to 3.5% 

(±1.5). A seed blend strategy and reduction in refuge from 20% to >2% represented a major 

paradigm shift for BPPD’s Insect Resistance Management (IRM) program; based on the 

recommendations of prior Scientific Advisory Panels (1998, 2000), the Agency had been 

discouraged from permitting seed mixtures as a refuge option for Lepidopteran pests due to 

potentially high movement of larvae between Bt and non-Bt plants. BPPD conducted a risk 

assessment (BPPD 2008a and 2008b) and presented preliminary conclusions to the Scientific 

Advisory Panel for peer review on February 23-24, 2009. The SAP (2009) expressed concern 

about the proposal to greatly reduce refuge for a single toxin, non-high dose PIP. The Panel 

concluded that there was no compelling evidence to support a reduction in refuge from 20% to 

>2% from the data provided by Pioneer or found in the public literature. However, the Panel 

agreed that a seed mix strategy would lend itself to corn rootworm biology. The final 

recommendation by the Panel was the use of a 20% seed blend for OAM1 due to the non-high 

dose characteristics of Herculex Cry34/35 corn. 

In 2009, Pioneer/DuPont amended its request from a 3.5% to a 5% seed blend refuge for OAM1. 

The Agency conducted two additional risk assessments in collaboration with EPA/ORD (BPPD 

2010a, 2010b), in which the BPPD IRM Team concluded that a 5% and 10% OAM1 seed blend 

- 4 - 
were significantly less durable as a 20% block refuge strategy. The Agency subsequently granted 

the applicant a time limited registration for a 10% OAM1 seed blend. 

 

This current BPPD risk assessment focuses on the seed blend portion for lepidoptera only. 

BPPD’s risk assessment conclusions for a 10% OAM seed blend for CRW were thoroughly 

presented and discussed in BPPD (2008b, 2010a, 2010b). 

II. PROBLEM FORMULATION 

The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) requires that registered 

pesticide uses do not pose unreasonable adverse effect to the environment. The purpose of this 

risk assessment is to provide insight into the potential effects to the environment as measured by 

the risk of resistance evolution in Bt corn target pests (lepidoptera) associated with the use of a 

5% seed blend registration for Optimum AcreMaxTM Insect Protection and 10% Optimum 

AcreMaxTM Xtra. 

A. Applicant Proposal 

Pioneer/DuPont requested to amend their current OAM registration by replacing the 5% 

structured refuge for lepidoptera with a 5% seed mixture and applying for a new stacked 

registration for OAM Xtra deployed as a 10% seed blend in the Corn Belt only. This risk 

assessment focuses on assessing the risk of resistance developing to a 5% OAM seed mixture in 

the main lepidoptera target pests, European corn borer (Ostrinia nubilalis, ECB) and 

southwestern corn borer (Diatraea grandiosella, SWCB). No separate modeling for the 10% 

OAM Xtra request was provided; hence the Agency could not assess the risk of resistance 

evolution to ECB and SWCB in a 10% OAM Xtra seed blend. To summarize, BPPD has 

reviewed the following elements of Pioneer’s proposal in this memorandum: 

Reviewed in this document:

• 5% lepidoptera seed blend (1507 x MON 810): biological, efficacy, and modeling data 

• 10% lepidoptera seed blend (1507 x 59122 x MON 810): biological and efficacy data 

only – no modeling submission 

Not Reviewed:

• 5% CRW seed blend: previously assessed in BPPD 2008b and BPPD 2010a and 2010b. 

- 5 - 
B. Seed Blend Literature 

This section includes a summary of BPPD’s literature search on the topic of seed blend literature, 

which is relevant to this risk assessment. 

Seed mixtures incorporating Bt and non Bt crop seeds have been a topic of discussion for 

approximately two decades. While such an Insect Resistance Management (IRM) strategy has 

obvious benefits (i.e., no grower compliance component, facilitating the planting of Bt and 

refuge fields), a variety of researchers have indicated that under some circumstances this 

approach could lead to more rapid evolution of resistance in some target pests (e.g., Mallet and 

Porter, 1992; Onstad and Gould, 1998; Davis and Onstad, 2000; and others). 

Scientific Advisory Panels (1998 and 2000) discouraged the Agency from the use of Bt seed 

mixtures to control lepidoptera target pest because substantial larval movement could be 

expected between Bt and non-Bt plants leading to more rapid selection of resistance. The SAP 

2011 also discouraged the use of pyramided PIPs deployed in seed blends for lepidoptera 

because of remaining uncertainties with larval movement and survival, especially for 

heterozygous genotypes (RS), and various unexplored epistatic effects. However, both the SAPs 

(2009 and 2011) determined that the seed blend strategy targeting corn rootworm would lend 

itself to the pest’s biology. 

Theoretical work by Mallet and Porter (1992) showed that insect resistance was accelerated in 

seed mixtures compared to Bt stands where 10% of the population was not exposed to selection, 

when the probability of larval movement ranged from intermediate to high (≥ 0.2), intensity of 

selection was great, and dominance of the resistance gene was low. They also reported that when 

insects selectively chose their food source (avoidance of toxic substances), then effective 

dominance was increased, and seed mixtures could delay resistance evolution but that predicted 

outcomes depended also on other circumstances. 

Using Mallet and Porter’s model, Tabashnik (1994) reported that 10% Bt seed mixtures were 

more durable than pure Bt stands and that block refuges of 10% were at least as durable (and 

more) as 10% seed mixtures when inheritance of resistance was recessive, partly recessive, or 

additive. As the percentage of refuge increased from 0-50%, Tabashnik further reported that a 

block refuge strategy performed equally well or better than seed mixtures. However, seed 

mixtures in conjunction with refuges were the most durable of all deployment strategies 

evaluated. 

Onstad and Gould (1998) recommended 20% block refuges adjacent to the Bt fields over seed 

blend strategies because of uncertainties surrounding expression of high-dose against the target 

pest. Their modeling results predicted that block refuges would be more durable than seed 

mixtures aimed at controlling lepidopteran pests. 

 

In a 2-yr field study with seed mixtures expressing Cry1Ab and non-Bt corn, Davis and Onstad 

(2000) obtained larval dispersal data, which were used to parameterize their simulation model 

and assess the durability of such a seed mixtures. Davis and Onstad observed that survival (after 

recovery) was lower for larvae that successfully dispersed from Bt to non-Bt plants compared to 

larvae that dispersed from non-Bt to non-Bt plants; this should favor heterozygote genotypes and 

- 6 - 
make inheritance of resistance less recessive (Mallet and Porter 1992). In addition, increased 

mortality of susceptible larvae moving off refuge plants onto Bt plants would result in a reduced 

effective refuge. They reported that neonate dispersal away from Bt plants was greater than 

dispersal observed from non-Bt plants and noted that extensive use of seed mixtures could select 

for populations with improved dispersal capabilities. The empirical data was used to 

parameterize the simulation model (Onstad and Gould, 1998), and it was determined that while 

seed mixtures delay evolution of resistance in ECB they are less effective at doing so than IRM 

strategies using a 20% block refuge. 

Carierre et al. (2004) concluded that the differences in Bt trait durability predicted by block 

refuges and seed mixtures in Mallet and Porter (1992) and Tabashnik (1994) were caused by a 

reduction in refuge insects in seed mixtures that had moved from non-Bt plants to Bt plants and 

could be overcome if the percent of non-Bt plants in seed mixtures was increased (compared to 

blocks). 

Onstad et al. (2011) discussed different IRM control strategies for corn pests. They concluded 

that seed mixtures make pest monitoring more difficult; IRM may be riskier because of larval 

movement and greater adoption of Bt crops; but block refuges themselves presented a risk 

because of lower compliance with IRM requirements. 

C. Uncertainties 

BPPD recognizes that to determine whether a 5% seed mixture for two pyramided lepidopteran 

controlling agents is as durable a strategy as the currently approved 5% block refuge for OAM, a 

number of uncertainties and questions need to be considered, some of which were also 

deliberated by the Scientific Advisory Panel (2011): 

1. Larval Movement

When susceptible ECB larvae move from Bt plants to non-Bt plants and their survival is lowered 

compared to susceptible larvae that have not previously been exposed to Bt (Onstad & Gould 

1998), then a seed mixture can be expected to decrease the effective refuge. Likewise, if 

susceptible larvae leave non-Bt plants, arrive on Bt plants, and are subsequently killed, then seed 

mixtures can be expected to reduce the refuge population to less than what can be expected to 

emerge from a block refuge. In either case, literature suggests that such conditions would 

increase the risk of resistance evolution in the target pests. It needs to be determined whether a 

reduction in effective refuge is substantial and could reduce the overall durability of the Bt 

product. 

There is evidence in the literature that 4th and 5th instar larvae of ECB move (White and Andow 

2007). Data from a two year study indicate that more than 50% of later instars move (White, 

personal communication). This sort of movement of heterozygous genotypes (RS) may lead to 

more rapid resistance evolution in the target pest. If toxins expressed in the Bt plants surrounding 

non-Bt plants express a high-dose against the target pest, then only truly resistant genotypes 

(RR) should survive and heterozygous individuals would die when moving. Such a scenario 

assumes that survival is multiplicative (not a conservative assumption), and that there is no 

epistasis between resistance loci. 

- 7 - 
2. Epistasis

The SAP (2010) discussed the importance of including assumptions of epistasis in the simulation 

model, and they specifically listed three types of mechanisms by which epistasis could manifest 

itself and resistance evolution could be sped up: a) low-level expression of Cry-proteases (more 

likely the case in species such as Helicoverpa zea), b) developmentally restricted expression of 

low levels of Cry-proteases (which may apply for late-instar ECB and SWCB), and c) crossresistance. 

3. Refuge Effectiveness in Seed Blends

ECB adults do not appear to detect the difference between Bt and non-Bt plants; however there is 

evidence that adult females discriminate between plants with and without damage. Additionally, 

in maize fields planted using a seed blend strategy, non-Bt plants can incur root damage from 

corn rootworm, which slows down plant growth. In such instances, the surrounding Bt plants 

with CRW protection (as in OAM Xtra) may grow faster and crowd-out the non-Bt plants. This 

ability to indirectly discriminate between Bt and non-Bt plants is a concern for seed blends rather 

than for block refuges. For first generation ECB laying their egg masses in a seed blend field 

expressing CRW PIPs, oviposition may be non-uniform. If there are no CRW traits expressed in 

the seed blend or no CRW infestations in the corn fields, the assumption that first generation 

ECB oviposit egg masses uniformly may be appropriate. For second generation ECB, the first 

described scenario above applies where non-random oviposition can be expected because 

females will choose undamaged (protected) plants rather than damaged (unprotected) plants. The 

net result is that greater neonate mortality can be expected for the second generation ECB (and 

first generation in CRW protected maize) in seed blends such as OAM resulting in fewer adult 

emergence compared to a structured refuge strategy with equal proportion of non-Bt plants. 

4. Effective Refuge

The SAP (2011) had concerns with the reduction in effective refuge that would result with a 5% 

seed blend relative to a 5% block refuge. Larval movement off refuge plants and decreased larval 

movement from surrounding Bt plants onto non-Bt plants may lead to a reduction of adults in 

seed blends compared to block refuges of equivalent non-Bt proportions. A reduction in refuge 

CEW would decrease the number of reverse migrants that could contribute their genes to the 

next generation of CBW in cotton growing regions, which may impact the natural refuge 

assumptions for products such as Bollgard II, WideStrike, and VipCot. A 5% seed blend with 

Optimum AcreMax grown in the Corn Belt would also, like SmartStax, contribute to a reduction 

in reverse migrating corn earworm. Hence the same concern applies to Pioneer/DuPont’s 5% 

seed blend product targeting lepidoptera. 

5. Mosaic of Bt Expression in Refuge Corn Ear

- 8 - 

Seed blends containing Bt and non-Bt seeds may actually accelerate resistance in ear feeding 

Lepidoptera including corn earworm and fall armyworm. For a 5% seed blend, 95% of the plants 

will be Bt and 5% will be non-Bt plants; refuge plants will have Bt plants as the dominant source 

of pollen. This pollen will contain post-segregation toxin alleles in the frequency of the parents 

(assuming no linkage). The effective dose of Bt encountered by larvae in these corn ears may not 

be as high as the surrounding Bt plants. Larvae that encounter this mosaic of toxins in the ears of 

refuge plants will not be neonates (likely second instar for CEW and FAW) and would require a 

larger dose of toxin to die. Bt ingestion has shown to promote wandering in larvae, and 

individuals that receive a sub-lethal dose may move to another kernel. This ability to move to 

another source of kernel in this mosaic of toxins and non-toxin environment will give 
heterozygous individuals a great fitness advantage; the functional dominance of the resistance 

allele will increase. None of the refuge plants in the seed blend field will be true-refuge plants to 

kernel feeders. (Porter 2011, personal communication). 

6. Focus of IRM Strategies

Work by Knutson and Gilstrap (1990) suggest that SWCB may have greater fecundity and larval 

survival than ECB. In addition, Ives and Andow (2002) showed in their model that greater net 

reproductive fecundity increases resistance evolution for PIPs that are high-dose. The SAP 

(2011) recommended for a pyramided cultivars an emphasis should be placed on durability for 

the pest that showed the greatest potential rate of resistance evolution. For seed blends and 

lepidoptera pests, this would likely be SWCB. 

III. PIONEER SUBMITTED STUDIES 

In this section, BPPD provides a summary of Pioneer/DuPont’s survival/movement and efficacy 

studies for different target pests and presents the applicant’s results and conclusions that were 

submitted to the Agency. 

A. European Corn Borer Survival 

Methodology: 

Pioneer/DuPont conducted a one year study in five locations across the Corn Belt to determine 

ECB larval survival from neonate to late instar when exposed to different seed blend percentages 

(5% and 10%) expressing a breeding pyramid of Cry1F and Cry1Ab (and stack with 

Cry34/35Ab1) or 100% refuge plots. The experimental set up was a randomized complete block 

design with 3 blocks. Artificial infestations were applied twice during the growing season and 

synchronized with the natural infestation timing of first and second generation ECB. Plants that 

were selected for sampling were dissected and live larvae were recorded. 

In the selected rows of seed blend blocks, each refuge plant and its next two consecutive Bt 

plants within the row were destructively sampled. In addition, three more Bt plants at least three 

plants away from the refuge plant were sampled (representing the pure stand portion of the 

blend) and investigated for surviving larvae. In the pure stand refuge plot, three non-consecutive 

plants were sampled, and numbers of live larvae were determined. 

A repeated measure analysis of variance was conducted using the square-root transformed 

averages and using the corresponding number of plants as weights. The main effect of treatment 

was analyzed as a randomized complete block structure with a random block effect. The main 

effect for plant type and the interaction effect between entry and plant type were modeled in the 

sub-plot level. Back transformed means and 95% CI for the average number of insects per plant 

were presented. The Cochran-Mantel-Haenszel test was used to study associations between instar 

and treatments in any of the three replications. 

Results: 

Manual infestations of first generation ECB did not establish well at any of the five locations 

based on the low recovery rate from refuge plants in all treatments (see Table 1). Recovery rates 

- 9 - 
in pure stand refuge plots ranged from 0.11 - 0.46 larvae/plant and in seed blend plots from 0 - 

0.58 larvae/plant. 

Recovery rate of second generation ECB was moderate and variable and ranged from 1.68 - 8.14 

larvae/plant in pure stand refuge plots and from 1.31 - 3.51 larvae/plant in seed blend plots. The 

recovery of larvae was significantly greater in pure stand refuge plots than 5% and 10% seed 

blends (50% of the time). In two locations, a 60% reduction of refuge insects was observed in 

seed blends. 

Table 1. Mean number of ECB late-instar larvae found on refuge plants in various treatments 

Location Refuge 

Blend Rate Plant Type 

ECB – G1 ECB – G2 

Number of 

Plants 

Sampled 

Mean 

Number of 

Larvae/Plant 

Number of 

Plants 

Sampled 

Mean 

Number of 

Larvae/Plant 

Johnston, IA 

5 

Refuge 

39 0 a - - 

10 77 0.01 a - - 

100 81 0.11 b - - 

Mankato, MN 

5 

Refuge 

39 0.31 ab 41 3.35 a 

10 73 0.18 a 45 2.75 a 

100 81 0.43 b 81 3.26 a 

Princeton, IL 

5 

Refuge 

38 0.58 a 40 3.11 a 

10 81 0.47 a 52 3.51 a 

100 81 0.46 a 81 8.14 b 

Windfall, IN 

5 

Refuge 

40 0.40 a 42 1.38 a 

10 77 0.23 b 81 1.65 a 

100 81 0.45 a 81 1.68 a 

York, NE 

5 

Refuge 

41 0.08 a 37 1.31 a 

10 74 0.26 b 73 1.57 a 

100 81 0.29 b 81 3.49 b 

Table was modified from original submission 

Statistical significance between treatments at each location provided after mean number of larvae/plant 

- 10 - 

Survival of first and second generation ECB on Bt plants in pure stands and seed blend plots 

(immediately adjacent or near refuge plant) was negligible and no significant differences 
between Bt treatments and distance away from refuge plants were noted (Table 2). The highest 

recovery rates for second generation ECB were observed in Bt plants adjacent to refuge plants 

and ranged from 0.1 to 0.13 larvae/plant. The limited numbers of larvae recovered were 

attributed to the likely minimal larval movement from refuge to protected Bt plants which 

expressed a high dose pyramid. 

Table 2 Mean number of first and second generation ECB larvae found on Bt plants adjacent to refuge plants compared 

to pure refuge stands 

Location Refuge 

Blend Rate Plant Type 

ECB – G1 ECB – G2 

Number of 

Plants 

Sampled 

Mean 

Number of 

Larvae/Plant 

Number of 

Plants 

Sampled 

Mean 

Number of 

Larvae/Plant 

Johnston, IA 

5 Bt 1 away 39 0 a - - 

5 Bt 2 away 39 0 a - - 

5 Bt pure stand 81 0 a - - 

10 Bt 1 away 77 0 a - - 

10 Bt 2 away 77 0 a - - 

10 Bt pure stand 81 0 a - - 

Mankato, MN 

5 Bt 1 away 39 0 a 41 0.01 a 

5 Bt 2 away 39 0 a 41 0 a 

5 Bt pure stand 81 0 a 81 0a 

10 Bt 1 away 73 0 a 45 0.01 a 

10 Bt 2 away 73 0 a 45 0 a 

10 Bt pure stand 81 0 a 54 0a 

Princeton, IL 

5 Bt 1 away 38 0.01 a 40 0.1 a 

5 Bt 2 away 38 0 a 40 0 b 

5 Bt pure stand 81 0 a 81 0 b 

10 Bt 1 away 80 0 a 52 0.11a 

10 Bt 2 away 80 0 a 52 0.12 a 

10 Bt pure stand 81 0 a 54 0 b 

Windfall, IN 

5 Bt 1 away 39 0 a 42 0 a 

5 Bt 2 away 40 0 a 42 0 a 

- 11 - 
5 Bt pure stand 81 0 a 81 0 a 

10 Bt 1 away 77 0 a 81 0.01 a 

10 Bt 2 away 77 0 a 81 0 a 

10 Bt pure stand 81 0 a 80 0.02 a 

York, NE 

5 Bt 1 away 41 0 a 37 0.02 a 

5 Bt 2 away 41 0 a 37 0 a 

5 Bt pure stand 81 0 a 81 0 a 

10 Bt 1 away 74 0 a 73 0.13 a 

10 Bt 2 away 74 0 a 73 0 a 

10 Bt pure stand 81 0 a 81 0.01 a 

Table was modified from original submission 

Statistical significance between treatments at each location provided after mean number of larvae/plant 

 

Pioneer/DuPont also compared the recovery rates of larvae/plant between refuge plants and 

immediately adjacent Bt plants in seed blends (Table not shown here). They found significant 

differences for first and second generation ECB at almost all locations. At four out of five 

locations, refuge plants in 5% and 10% seed blends had significantly more larvae (95% C.I.) than 

immediately adjacent Bt plants. Generally, for first generation ECB, very few larvae were 

detected on Bt plants (range 0.0-0.01 larvae/per plant), while some larvae were found on refuge 

plants (range 0.08-0.58 larvae/plant). For second generation ECB, somewhat more larvae were 

detected on Bt plants than for first generation ECB (range 0.0-0.13). Second generation 

establishment on refuge plants ranged from 1.31-3.35 larvae/plants. 

Pioneer/DuPont Conclusions: 

“Data from this study show that deployment of 1507 x 59122 x MON810 maize with blended 

refuge as low as 5% can produce susceptible ECB at rates comparable to pure stand refuge. 

However, insect production from blended refuge plants was significantly less than insect 

production from pure stand of refuge 50% of the time for second generation ECB. The results 

collectively suggest that movement between Bt and non-Bt plants in blended refuge is minimal, 

and that protection against both first and second generation ECB remains excellent when 

deploying 1507 x 59122 x MON810 maize in seed blends.” 

- 12 - 
B. Southwestern Corn Borer Survival

Methodology: 

Pioneer/DuPont conducted a study at one location each in Kansas and Tennessee in 2009 to 

determine SWCB larval survival from neonate to late instar when exposed to different seed blend 

percentages (5% and 10%) expressing a breeding stack of Cry1F and Cry1Ab (as well as 

Cry34/35Ab1) or 100% refuge plots. This experiment relied on natural SWCB infestations; 

otherwise, the same methodology was used as described earlier in section III.A.1. 

Results: 

Natural infestations were low in both locations based on the recovery rates of late-instar larvae 

from pure stand refuges and refuge plants from seed mixture plots. Recovery rates were 0.03 

larvae/ plant in refuge plots and ranged from 0.01-0.03 larvae/ refuge plants in seed blends at 

Garden City (KS). At Union City (TN), recovery rates from refuge plots were 0.41 larvae/ plant 

and ranged from 0.28-0.26 larvae/refuge plant in seed blends. Significant differences were only 

observed between treatments at Union City. 

Survival on pure stand Bt and Bt plants adjacent to refuge plants in 5% and 10% seed blends was 

negligible and near 0. No significant differences between treatments were noted. 

Survival on refuge plants and immediately adjacent Bt plants in 5% and 10% seed blends was 

significantly different between treatments at both locations. At Garden City (KS), for example, 

0.01 larvae/refuge plant in 5% seed blend plots and no survivors on adjacent Bt plants were 

noted. Survival in refuge plots was significantly higher than on Bt plants (distant to refuge plant) 

in seed blends. 

Pioneer/DuPont Conclusions: 

“Data from this study show that deployment of 1507 x 59122 x MON810 maize with blended 

refuge as low as 5% can produce susceptible SWCB. The results suggest that blended refuge 

plants do serve as a functional SWCB refuge, although at one location blended refuge produced 

less susceptible insects than pure stand refuge plants.” 

C. Efficacy of 1507 x 59122 x MON810 Maize Against ECB 

Methodology: 

In 2009, the efficacy of 1507 x 59122 x MON810 maize against ECB was evaluated at five US 

locations (York (NE), Johnston (IA), Mankato (MN), Ivesdale (IL), and Windfall (IN)). At each 

location the experimental set up was a randomized complete block design with three blocks and 

three treatments (pure stand 1507 x 59122 x MON810 maize, 95% seed blend with 1507 x 59122 

x MON810 with 5% near isoline maize, and pure stand 59122 maize (refuge plot for ECB)). The 

- 13 - 
5% isoline maize was included to simulate a seed blend for ECB, SWCB, and CRW. Plants were 

artificially infested at two time intervals during the growing season to simulate infestation from 

1st and 2nd generation ECB. Injury from first generation ECB leaf feeding was recorded using a 9 

to 1 visual rating scale where a score of 9 indicated ‘no injury’ and a score of 1 indicated ‘heavy 

injury’. The visual rating scale was similar to that published by Guthrie et al. (1960) with 

numbers in the reverse order. Injury from second generation ECB feeding was evaluated 

measuring the amount of stalk tunneling in centimeters. The experimental unit for this study 

consisted of a plot of maize eight rows wide and 52.5 ft in length. Four five-plant clusters in each 

of the three treatments were identified for measuring ECB efficacy. 

Analysis of variance was used to analyze main effects and Fisher’s protected LSD test was used 

to determine differences among least squares means when appropriate. Three separate analyses 

were conducted on the efficacy data: comparison between 1) ECB injury to refuge plants in 5% 

blends and refuge plants in pure stand isoline plots; 2) efficacy of 1507 x 59122 x MON810 

plants adjacent to Bt plants next to refuge plants in a 5% seed blend and pure stand Bt plants; 3) 

efficacy of Bt plants adjacent to Bt plants next to refuge plants in a 5% seed blend. 

Results: 

In four out of five locations, leaf injury to refuge plants from first generation ECB was 

significantly greater (the greater the damage, the smaller the assigned score) in pure 1507 x 

59122 x MON 810 plots (range of ratings 6.2-7.5) than in seed blend plots (range of ratings 7.2-

8.3). Similarly, stalk tunneling damage was greater (the greater the damage, the greater the 

assigned score) in refuge plants from pure stand plots (4.6-18.1 cm) than in refuge plants from 

seed blend plots (range 1.9-9.4 cm). In two out of three locations, these observed differences 

between treatments were statistically significant (data tables not shown but were provided in 

MRID 480707-02). 

When the efficacy was compared between Bt plants in seed blend (adjacent and two plants 

removed from refuge plant) and Bt plants from pure stand plots, there were no significant 

differences noted in leaf injury caused by first generation ECB. In addition there were no 

significant differences in damage noted between Bt plants from seed blend plots (adjacent and 

two plants removed from refuge plant). Similarly, stalk tunneling damage caused by second 

generation ECB was not significantly different between Bt plants from pure stand and seed blend 

plots, as well between Bt plants (adjacent and two plants removed from refuge plant) from seed 

blend plots (data tables not shown here but were provided in MRID 480707-02). 

Pioneer/DuPont Conclusions: 

“The 1507 x 59122 x MON810 maize plants in close proximity to blended refuge plants will be 

at low risk to economically significant reductions in efficacy against ECB. Refuge plants 

deployed in a seed blend with 1507 x 59122 x MON810 maize showed similar to or less ECB 

damage than refuge plants in separate pure stand plantings. Some damage observed to refuge 

plants in seed blends indicates, however, that refuge plants deployed in seed blends with 1507 x 

59122 x MON810 maize should serve as a functional ECB refuge.” 

- 14 - 
D. Efficacy of 1507 x 59122 x MON810 Maize Against ECB 

Methodology: 

In 2009, the efficacy of 1507 x 59122 x MON810 maize against SWCB was evaluated at three 

US locations (Garden City (KS), West Memphis (AR), and Union City (TE)). The study relied 

on natural pest infestations, yet the experimental design and statistical analyses were the same as 

those described in the methodology section for ECB (Section III.B.1) (however, no leaf damage 

determination). Stalk tunneling damage was determined by splitting the lower eight internodes of 

each corn stalk sampled and recording the total amount of tunneling per plant in centimeters. 

Results: 

No statistically significant damage was observed between refuge plants in seed blends and pure 

stand plots. Damage from stalk tunneling in seed blends ranged from 2.4-5.1 cm and from 3.6-

6.2 cm in 100% refuge treatments. No damage was observed on Bt plants in pure stands and seed 

blends (adjacent to and two plants removed from refuge plant), and hence, no significant 

difference were observed between the treatments as well as between Bt plants from seed blends 

at all locations. 

Pioneer/DuPont Conclusions: 

“The 1507 x 59122 x MON810 maize plants in close proximity to blended refuge plants will be 

at low risk to economically significant reductions in efficacy against SWCB. Injury observed to 

refuge plants indicates that refuge plants deployed in seed blends with 1507 x 59122 x MON810 

maize should serve as a functional SWCB refuge.” 

1. Fall Armyworm

Methodology: 

The efficacy of OAM (1507 x MON810) and OAM Xtra (1507 x 59122 x MON810) was 

evaluated at three locations in Iowa (Johnston, Earlham, and Marion) during the 2009 corn 

growing season. The experimental design was a randomized complete block design with 3 blocks 

and several treatments with three replicates each. The treatments relevant to this review were 

100% OAM Xtra, 100% non-Bt, and seed blends using two different hybrid seeds, 95% OAM 

and OAM Xtra with 5% non-Bt. Five plants in five plant clusters each (25 plants/plot) were 

artificially infested with 100 neonates /plant at the first infestation and 25 neonates/plant at the 

second infestation date (except at Johnston, where larvae from first infestation did not establish, 

and 100 larvae/plant were applied at the second infestation date); feeding damage was visually 

assessed three weeks after infestation using a 9 to 1 rating scale where “9” indicates “no 

damage” and “1” indicates “heavy injury”. The experimental unit in this experiment consisted of 

a block that was four rows wide and 17.5 ft long. Efficacy of all treatments was assessed in a five 

plant cluster in rows 3 and 4. 

- 15 - 
Analysis of variance was used to analyze main effects and Fisher’s protected LSD test was used 

to determine differences among least squares means when a significant main effect occurred. 

Three comparative analyses were conducted of FAW damage: 1) between refuge plants in seed 

blends and 100% non-Bt plots, 2) between Bt plants (adjacent and two plants removed from 

refuge plant) in seed blends and 100% Bt plots, and 3) between Bt plants adjacent and two plants 

removed from refuge plant in seed blends. 

Results: 

No statistically significant difference in leaf damage from FAW was detected on refuge plants in 

5% OAM Xtra seed blends compared to 100% refuge plots tested in three different locations. 

Leaf damage on refuge plants in seed blends ranged from 4.9-7; damage in the 100% refuge plot 

ranged from 4.1-7.2. Refuge plants in OAM plots suffered less damage (range of ratings 6.2-7.5) 

than plants in 100% refuge plots (5.0 – 7.2). These differences were statistically significant at 

two out of three locations. The applicant attributed these differences in damage on the two types 

of hybrids (OAM and OAM Xtra) to initial neonate dispersal from refuge plants to surrounding 

Bt plants in OAM seed blends. 

When the efficacy was compared between Bt plants from the two hybrid seed blends (adjacent 

and two plants removed from refuge plant) and the 100% OAM Xtra plots, no statistically 

significant differences were found for OAM Xtra seed blends. For OAM seed blends at one out 

of two sites, the damage to Bt plants in seed blends was greater (mean of 8.9±0.5) compared to 

the pure stand Bt (mean of 9.0±0.00). When the efficacy was compared between Bt plants 

adjacent and two plants removed from refuge plants, no statistically significant differences were 

found. Damage ratings pooled for both hybrids ranged from 8.8 – 9.0 for plants immediately 

adjacent to refuge plants and were 9.0 for those that were two plants removed. 

Pioneer/DuPont Conclusions: 

“The 1507 x MON810 or 1507 x 59122 x MON810 maize plants in close proximity to blended 

refuge plants will be at low risk to economically significant reductions in efficacy against FAW. 

Refuge plants deployed in a seed blend with either 1507 x MON810 or 1507 x 59122 x MON810 

maize showed significant less FAW injury in 33% of the location by hybrid comparisons. In the 

remaining 67% of the comparisons, there were no significant differences between refuge plants 

deployed in seed blends and refuge plants in pure-stand plantings. 

2. Corn Earworm

Methodology: 

In this study, Pioneer/DuPont evaluated the efficacy of Optimum AcreMax Xtra (Cry1Ab, 

Cry1F, and Cry34/35) against corn earworm in five field plots in Johnston (IA), Earlham (IA) 

with two sites, and Marion (IA) with two sites. The experiment was conducted as a randomized 

complete block design. The three treatments were 100% OAM Xtra, 100% refuge (control), and 

a seed blend with 95% OAM Xtra and 5% refuge seed. 

- 16 - 
Artificial infestations with neonate CEW were carried out with approximately 65 

individuals/corn ear-per plant. Each treatment had 5 identified plant clusters that were chosen for 

infestation purposes. Injury from CEW feeding was scored approximately three weeks after the 

infestation date and assessed by measuring the total square centimeters of damage on ear tips and 

kernel area. Additionally, the number of live CEW found in a five-plant cluster was recorded at 

the time of scoring. 

ANOVA was used to analyze the main effects and Fisher’s protected LSD test to determine 

differences among LSM as appropriate when a significant main effect occurred. 

Results: 

When CEW injury was compared between refuge plants from seed blend plots and pure stand 

isoline plots, no statistically significant difference in damage was reported for three out of five 

plots. In two plots, the damage incurred on refuge plants from seed blend was lower than on 

plants from pure stand refuge plots (statistically significant). Damage in seed blends ranged from 

2.8 to 13.4 cm2/plant. Damage in refuge plants from isoline plots ranged from 5.1 to 17.0 

cm

2

/plant. The difference in larvae/ear from the two types of refuge plants was not statistically 

significant, and mean numbers ranged from 0 to 0.4/ear in seed blends and 0.1 to 0.3/ear in 

isoline plots. 

Furthermore, the efficacy of the Bt plants surrounding the refuge plant in seed blends 

(immediately adjacent and two plants removed) was compared to the efficacy of Bt plants in 

pure stand Bt plots. No significant differences between the different types of Bt plants were 

detected. The mean number of larvae/ear ranged from 0 to 0.6 in seed blends and from 0.1 to 0.7 

in pure stand Bt plots. 

When damage and survival were compared between the Bt plants (adjacent and two plants 

removed from refuge plant) in seed blends, no statistically significant differences were detected. 

Damage ranged from 0.1 to 2.0 cm2/plant, and mean larvae/ear ranged from 0 to 0.6. 

Pioneer/DuPont Conclusions: 

“Data from this study show that maize containing a pyramid of 1507 and MON810 provided 

very good efficacy against moderate to high manual infestations of CEW. These results also 

demonstrate that OAM Xtra maize plants in close proximity to refuge plants in a seed blend will 

be at low risk to economically significant reductions in efficacy against CEW. Refuge plants in a 

seed blend showed significantly less CEW damage at two of the five locations. At the remaining 

three locations, there were no significant differences between refuge plants deployed in seed 

blends and refuge plants in pure stand plantings. Additionally, these results demonstrate that 

OAM Xtra plants deployed with a 5% refuge are similar in efficacy to OAM Xtra maize plants 

planted in a pure stand.” 

A. Pioneer/DuPont Modeling Analysis for ECB and SWCB 

- 17 - 
In this section, BPPD summarizes Pioneer/DuPont’s model structure, modeling scenarios tested, 

assumptions, parameter values, and the applicant’s results and conclusions that were submitted to 

the Agency. 

Model Structure: 

Pioneer/DuPont’s model was deterministic, spatially explicit, and consisted of a grid 100 m x 

100 m cells (each cell = one hectare); simulations occurred on 400 cells for a total of size of 400 

ha. The grid was divided into 10 fields (4 x 10 cells, 40 ha), and each cell within each field was 

assigned to Bt, non-Bt, or Bt corn with a 5% blended refuge. The structured refuge was 

simulated by assigning two of the 40 cells in a field to non-Bt corn (see Figures 1 and 2). The 

longest distance between refuge areas of each field was limited to < 600m; this placement 

favored block refuges and assumed random mating for ECB. Block refuges were all placed on 

the same short side of the ten rectangular fields and kept fixed at that location year after year 

(modeling scenario A) or switched to the opposite side each consecutive year (modeling scenario 

B). To simulate a 5% blended refuge, all 40 cells in each field were designated to have 5% nonBt maize. Simulations were run for 400 generations (200 years). A torus was simulated so that 

adults moving off of one side of the system arrived on the other side. 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

133333333333333333333 100330033003300330033

233333333333333333333 233333333333333333333

33333

33333

33333

33333

33333

33333

33333

33333

03300

33333

33333

33333

33333

33333

33333

33333

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33333

333333333333333333333 3333333333333333

433333333333333333333 4333333333333333

533333333333333333333 5333333333333333

633333333333333333333 6333333333333333

733333333333333333333 7333333333333333

833333333333333333333 8333333333333333

933333333333333333333 9333333333333333

10 3 3 0 0 3 3 0 0 3 3 0 0 3 3 0 0 3 3 0 0 10 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

11 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 11 3 3 0 0 3 3 0 0 3 3 0 0 3 3 0

12 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 12 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

13 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 13 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

14 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 14 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

15 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 15 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

16 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 16 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

17 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 17 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

18 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 18 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

19 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 19 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

20 0 0 3 3 0 0 3 3 0 0 3 3 0 0 3 3 0 0 3 3 20 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Figure 1. 5% block refuge placement in uneven years; Figure 2. 5% block refuge placement in even years 

(Schematic reproduced from MRID480707-01; 0=cell with refuge plants; 3=cells with 1507 x MON810 plants) 

Model Scenarios: 

Scenario A: 5% block refuge with refuge in a fixed location 

Scenario B: 5% block refuge with refuge moved yearly (see Figure 1 above) 

Scenario C: 5% blended refuge 

- 18 - 
Assumptions and Parameters: 

Pioneer/DuPont evaluated the durability of OAM by investigating a set of worst-case as well as 

benchmark parameters for European corn borer and southwestern corn borer (see Table 3). 

Benchmark parameters represented what the applicant assumed to be the most likely estimates 

and which were supported by direct measurements, reports in the literature, or communications 

from academic experts. Worst-case parameters represented values of uncertainty for which there 

was less empirical support or published data were scarce. 

The applicant stated that dominance values were estimated based on empirical data testing of 

homozygous susceptible and resistant, as well as heterozygous resistant insects from Cry1F and 

Cry1Ab resistant colonies. It was assumed that there were two resistant loci, one for Cry1Ab 

with alleles A (susceptible type) and X (resistant type), and one for Cry1F with alleles B 

(susceptible type) and Y (resistant type). Resistant individuals (YY and XX) had 100% survival, 

and there was no cost to resistance incorporated into the model. All other genotypic survival 

rates were derived from empirical data or published literature. Other survival parameters were 

incorporated such as egg-to-neonate survival rate (0.9025), pre-dispersal tasting, and overall 

survival to the late instar stage, carrying capacity, as well as overwintering survival. 

Benchmark values for cross-resistance were set at zero survival on Cry1Ab for ECB and SWCB 

individuals that were resistant to Cry1F. ECB and SWCB that were resistant to Cry1Ab were 

assumed to have 0.2 and 0.1 survival on Cry1F. 

Pioneer/DuPont declared resistance (product failure) in the simulations when the resistance allele 

frequencies (X and Y) exceeded 0.5 for both traits, and the number of years of durability was 

reported when this defined threshold in the model was exceeded. 

Adult ECB were allowed to disperse before (random) mating took place. For SWCB, only 1% of 

males were allowed to move before mating occurred; most mating was assumed to take place in 

the natal fields. The dispersal range for both species was set at one square mile (17 x 17 cells); 

and uniform distribution of adults was assumed after the dispersal event. Female post-mating, 

pre-ovipositional dispersal was modeled at 100% for ECB and 25% for SWCB, and one square 

mile was used as the default dispersal area. 

Table 3. Various Modeling Parameters and Values used in Pioneer/DuPont's Simulations 

Parameter 

European Corn Borer Southwestern Corn Borer 

Benchmark Worst-Case Benchmark Worst-Case 

Cross-resistance (survival) of: 

(1) Cry1Ab to Cry1F 

 (2) Cry1F to Cry1Ab 

0.2 

0 

0.3 

0.1 

0.1 

0 

0.3 

0.1 

IRA frequency for Cry1F 0.01 0.05 0.001 0.005 

IRA frequency for Cry1Ab 0.001 0.005 0.001 0.005 

- 19 - 
Parameter 

European Corn Borer Southwestern Corn Borer 

Benchmark Worst-Case Benchmark Worst-Case 

Dominance of resistance alleles 0.01 0.05 0.02 0.1 

Coefficient of dominance (fold 

difference between 1st and late instars) 2 fold 2 fold 

Coefficient of dominance (fold 

difference between vegetative and 

reproductive corn) 

2 fold 2 fold 

Pre-dispersal tasting survival 0.6 0.95 

Dominance of pre-dispersal tasting 

survival 0.01 0.05 0.02 0.1 

SS survival on Cry1Ab 

(1) First instar 

(2) Late instar 

0.001 

0.025 

0.01 

0.2 

0.001 

0.05 

0.01 

0.2 

SS survival on Cry1F 

(1) First instar 

(2) Late instar 

0.001 

0.025 

0.01 

0.2 

0.001 

0.25 

0.01 

0.75 

RR survival on Cry1F and Cry1Ab 1 1 1 1 

Oviposition rate (eggs/female) 288 441 

Probability of 1st instar leaving refuge 

plant 0.76 0.76 

Probability of 1st instar leaving Bt plant 0.98 0.98 

Carrying capacity in vegetative plant 

(first generation) 18 1 

Carrying capacity in reproductive part 

of plant (second generation) 22 1 

Overwintering survival rate 0.18 0.14 

Male and female dispersal range 800 m 800 m 

Proportion of eggs produced in natal 

field 0 0.75 

Egg production dispersal range 800 m 800 m 

Egg dispersal distribution Uniform Uniform 

This table was modified from the original submission (MRID 480707-01) and does not include all parameters reported by the 

applicant. 

- 20 - 
Model Verification: 

The values in the matrices set up by the model were verified by manual calculation. The verified 

matrices included the dominance value, cross resistance, mating frequency, survival rate, and 

oviposition. 

Results from the current Pioneer/DuPont model were verified against Pioneer’s model used to 

support a reduction in refuge for MON810 x 1507 (MRID 480056-02). Several key parameters 

were held constant in both models during this process. The predicted durability for block refuges 

was consistent between both models. 

The underlying logic of the program was tested by running extreme scenarios such as 100% 

resistance allele frequency, no resistance genes, 100% Bt maize, and 100% refuge maize for both 

models; results from the two models were similar. 

Additionally, several scenarios modeled by Guse et al. (2001) were also run using their 

parameter sets in Pioneer/DuPont’s model. The results between the two models were 

comparable, which indicates that the model was functioning properly. 

Because of the high-dose nature of toxins in 1507 and MON810 against ECB and SWCB, the 

pest populations decreased each year. In many simulations, the population reached a level at 

which the density of adults was so low that reproduction became unlikely and local extinction 

was approached. Extinction or extirpation was defined by Pioneer/DuPont as “the number of 

years it took for the overall adult density to go below one adult/hectare”. 

Sensitivity Analyses: 

The purpose of this analysis was to determine which parameters had the largest impact on the 

model predictions by varying one parameter (or a related set of parameters) at a time while 

holding all others constant. 

Sensitivity analysis for ECB and SWCB were conducted for the several parameters displayed in 

Table 4 below. A parameter was determined to be sensitive when a change in its value resulted in 

less than 200 years durability of 1507 x MON810. In addition to years of durability, years for 

local populations to approach extirpation (<1 individual/ha) before reaching the durability 

threshold (resistance allele frequency = 0.50) were calculated. If a scenario predicted extirpation, 

the model still estimated time to resistance evolution. 

Table 4. Sensitivity analysis conducted for some parameters used in ECB and SWCB modeling simulations 

Parameter Range for 

Parameter values Sensitivity 

ECB 

Initial adult density 5,000-50,000 Durability >200 yrs; no 

- 21 - 
Range for Parameter Sensitivity Parameter values 

Plant density 52,611-87,685 sensitivity apparent. 

Extirpation occurred within 2-

Coefficient of dominance (fold difference 4 years. 

between vegetative and reproductive maize) 1-5 fold 

Pre-dispersal tasting survival 0.4-0.7 

Survival of susceptible 1st instar leaving Cry1Ab 

or Cry1F 0.0001-0.05 

Probability of 1st instar leaving Bt 0.7-0.99 

Probability of 1st instar leaving refuge plant 0.4-0.9 

1st instar survival while dispersing 0.1-0.4 

Proportion of males and females mating in natal 

fields 0-0.5 

Proportion of eggs produced in natal fields 0-0.5 

Cross-resistance of Cry1Ab to Cry1F 0.1-0.7 

Sensitivity apparent at high 

values. 

Extirpation occurred in 3 yrs 

for all values/scenarios 

modeled. 

Cross-resistance of Cry1F to Cry1Ab 0-0.3 

Initial resistance allele frequency X 0.0005-0.01 

Initial resistance allele frequency Y 0.005-0.1 

Dominance of resistance alleles 0.001-0.2 

Dominance for pre-dispersal tasting survival 0.001-0.2 

Survival rate of susceptible late instars on 

Cry1Ab 0.005-0.5 

Block refuge durability 

>200yrs for all values; no 

sensitivity apparent. Survival rate of susceptible late instars on Cry1F 0.005-0.5 

Probability of late instars leaving non-Bt or Bt 

plants 0.025-0.25 Seed blend durability was sensitive at high end of values 

Late instar survival rate while dispersing 0.25-0.90 

For both block and blend, 

Coefficient of dominance (fold difference Extirpation occurred in 3yrs. 

between 1st and late instar stage) 2-10 fold 

Blend percentage 0.01-0.1 Sensitivity al low end; 

extinction in 1.5-5 yrs 

Parameter Range for 

Parameter values Sensitivity 

- 22 - 
Range for Parameter Sensitivity Parameter values 

SWCB 

Initial adult density 700-5,000 

Durability >200 yrs; no 

sensitivity apparent. For 5% 

block refuge with fixed 

location, no extirpation was 

reached within the time 

modeled. For 5% block with 

refuge moved yearly, 

extinction occurred in 5.5 

years. For 5% seed blend, 

extinction occurred in 4 years.

Plant density 52,611-87,685 

Coefficient of dominance (fold difference 

between vegetative and reproductive maize) 2-15 fold 

Pre-dispersal tasting survival 0.9-1 

Survival of susceptible 1st instar leaving Cry1Ab 

or Cry1F 0.0001-0.05 

Probability of 1st instar leaving Bt 0.7-0.99 

Probability of 1st instar leaving refuge plant 0.4-0.9 

1st instar survival while dispersing 0.1-0.4 

Proportion of males and females mating in natal 

fields 0.5-1 

Proportion of eggs produced in natal fields 0.5-0.9 

Cross-resistance of Cry1Ab to Cry1F 0.05-0.7 Model showed sensitivity at 

high end values modeled for 

blended refuge and block with 

refuge location moved yearly. 

For 5% block (fixed refuge), 

no extinction occurred within 

the timeframe modeled. When 

the refuge was moved, 

extinction occurred in 5.5 

years. For blends extinction 

occurred in 4 years. 

Cross-resistance of Cry1F to Cry1Ab 0-0.3 

Initial resistance allele frequency X 0.0005-0.01 

Initial resistance allele frequency Y 0.005-0.1 

Dominance of resistance alleles 0.001-0.2 

Dominance for pre-dispersal tasting survival 0.001-0.2 

Survival rate of susceptible late instars on 

Cry1Ab 0.005-0.5 For block refuge, no 

sensitivity was apparent. 

Extinction occurred in 5.5 

years when refuge was moved. 

For the blend, sensitivity was 

apparent at high end of values 

and extinction occurred in 4 

years. 

Survival rate of susceptible late instars on Cry1F 0.05-0.75 

Probability of late instars leaving non-Bt or Bt 

plants 0.025-0.25 

Late instar survival rate while dispersing 0.25-0.90 

Blend percentage 0.01-0.1 

Durability > 200 years for all 

values for blended refuge. 

Model not sensitive to range 

of values and modeling 

horizon tested. Extirpation 

occurred in 2-11 years. 

This table was modified from the original submission (MRID 480707-01) 

- 23 - 
Results of Sensitivity Analysis: 

European corn borer: 

For structured refuge (fixed or moved location) and seed blend modeling, the following 

parameters were determined to be sensitive (an apparent reduction in durability): initial 

resistance allele frequency, dominance, and cross-resistance. In addition to these three 

parameters, late instar survival and movement, blended refuge percentage were also determined 

to be sensitive in seed blend simulations. 

Southwestern corn borer: 

For structured refuge simulations, it was relevant whether the refuge locations were held fixed or 

moved yearly. Less sensitivity to parameters was observed when refuge locations remained 

unchanged. The applicants attributed the lack in sensitivity to the biology of the pest, which 

typically mate and oviposit in natal fields. Also, durability was greatly extended when refuge 

was held fixed because this allowed the susceptible population to build up year after year. For 

block refuge simulations with the refuge location moved yearly, cross-resistance, initial 

resistance allele frequency, and dominance were sensitive parameters. 

For seed blends, cross-resistance, initial resistance allele frequency, dominance, coefficient of 

dominance on reproductive maize, late instar movement and survival, and blended refuge 

percentage were deemed sensitive parameters. 

Results of Modeling Scenarios: 

Using benchmark parameter assumptions, both the structured refuge and seed blend strategy 

were predicted to have a greater durability than 200 years for European corn borer and 

southwestern corn borer (resistance assumed when RAF >0.5). For ECB worst-case scenarios, 

block refuges had a durability of approximately 35 years, while seed blends had a durability of 

26 years. The durability for block refuges was greater than for seed blends because of effects of 

late instar survival and larval movement in seed blends. Results are displayed in Table 5 below. 

For SWCB, with best-case assumptions the predicted durability for 5% block and seed blend was 

again >200 years. Using worst-case assumptions, block refuges had a predicted durability of > 

200 years when refuges were held fixed each year; when refuges were moved yearly, then the 

durability decreased to just 11 years. This change in durability was attributed to the mating 

behavior of SWCB which has been shown to occur primarily in the native field. A structured 

refuge that is maintained in the same location year after year would then create a sink of 

susceptible insects over time. However, the applicant contended that based on their unpublished 

data growers who planted CRW traits were more likely to move refuges rather than keeping 

them in the same location year after year. The point of population extinction was reached by 5.5 

years when refuges were moved yearly. Seed blends were predicted to have a durability of 16 

years under worst-case assumptions. 

- 24 - 

Pioneer/DuPont stated that a key consideration was whether refuge plants in a Bt seed blend 

produced sufficient amounts of susceptible insects to effectively delay resistance evolution. The 
applicants concluded that the model accurately predicted a reduction in “effective refuge” from a 

seed blend (lower production of susceptible insects) compared to a block refuge of equivalent 

non-Bt proportion but that the number of insects emerging from refuge plants was sufficient to 

provide a durability of >200 years for a 5% OAM seed blend. 

Table 5. Estimated durability for a 5% OAM seed blend and 5% OAM structured refuge 

Refuge Type 

European Corn Borer Southwestern Corn Borer 

Benchmark 

(yrs to 

resistance) 

Worst-Case 

(yrs to 

resistance) 

Benchmark 

(yrs to 

resistance) 

Worst-Case 

(yrs to 

resistance) 

5% Block 

(location fixed) 

Durability >200 36 >200 >200 

Extirpation 3 3.5 N/A N/A 

5% Block 

(moved yearly) 

Durability >200 35 >200 11 

Extirpation 3 3.5 N/A 5.5 

5% Seed Blend 

Durability >200 26 >200 16 

Extirpation 3 3 4 4 

This Table was reproduced from MRID 480707-01// 1 Durability is the relative year when the resistance allele frequency exceeds 

50% for both traits; 2

 Extirpation is the number of years it takes for the adult density to go below one adult/hectare 

Pioneer reported that as populations approached extirpation that the predicted durability 

exceeding that estimate should be considered with caution because random genetic drift may 

have a stronger influence on gene frequency than selection, and that random mating along with 

other assumptions may be violated. 

Pioneer/DuPont Conclusions: 

“Maize containing events 1507 x MON810 deployed with a 5% blended refuge is predicted to 

have >200 years of durability for ECB and SWCB. A 5% blend produces sufficient susceptible 

insects to significantly extend the durability of this trait combination. The pyramid of two highdose traits in 1507 x MON810 effectively mitigates the risk that was historically associated with 

blending lepidopteran refuge for single trait high-dose products. The approval of a blended 

approach to refuge deployment will significantly reduce the complexity of planting and 

management of refuge acres and will have a positive impact on refuge compliance. Harmonizing 

refuge deployment for stacked lepidopteran and corn rootworm Bt products ensures that refuge is 

placed appropriately for these traits each and every time a bag of Bt seed is planted, especially 

important for non-high dose corn rootworm traits. Blended refuge benefits not only the grower 

(by reducing planting complexity and refuge management) but the public as well, by ensuring 

100% compliance, thereby extending the lifespan of more environmentally friendly technology.” 

- 25 - 
IV. BPPD REVIEW 

A. Biological data 

Pioneer/DuPont conducted a study to determine ECB and SWCB larval survival in seed blends 

expressing the pyramided PIP OAM. The applicant relied on artificial ECB infestations, yet the 

number of (simulated) first generation larvae establishing successfully on refuge plants in pure 

plots as well as in seed blends was low. Sometimes those differences were significant, other 

times they were not. Mean numbers of larvae/plant for the (simulated) second generation were 

much greater however than for the first generation - sometimes by 20 fold (see Table 1). It is 

unclear what affected the establishment of first generation larvae on refuge plants in this trial. 

BPPD concludes that these reported establishment numbers are preliminary data and that further 

trials should be conducted to gather additional data (multi-year, multi-location study) to more 

accurately estimate larval establishment on refuge plants in seed blends and pure stands maize. 

BPPD concludes that the establishment ECB numbers on refuge plants in seed blends from 

second generation infestation trials likely represent an overestimate because second generation 

adults may be less likely to oviposit onto refuge plants in 5% and 10% seed blends than they 

would otherwise do in a structured refuge configuration. The SAP (2010) stated that while ECB 

adults cannot distinguish between Bt and non-Bt expressing plants per se, second generation 

adults recognize damage on refuge plants from the prior generation and may likely choose to 

oviposit onto non-damaged (Bt protected) corn plants in seed blends. Additionally, refuge plants 

that incurred corn rootworm damage in seed blends could easily be crowded out by Bt 

protected/surrounded plants which would also lead to ECB adults to selectively oviposit more 

likely onto Bt plants than refuge plants. BPPD concludes that the estimated number of adults 

emerging from seed blends, as reported by Pioneer/DuPont, could in some cases be even lower 

than 60% (max. estimated) compared to emergence in the block refuges; such incidents could 

occur more than 50% of the time (as reported by the applicant). 

Pioneer/DuPont reported that mean ECB larvae (1st gen) on Bt plants surrounding refuge plants 

in seed blends was 0 insects/plant (see Table 2). This is not surprising considering that 

establishment of larvae on refuge plants was low. Establishment for 2nd generation ECB on 

refuge plants in seed blends was greater, and hence, the n umber of larvae encountered on 

surrounding Bt plants increased correspondingly. BPPD concludes that these preliminary data 

are not sufficient to draw final conclusions about larval movement and survival from refuge 

plants onto surrounding Bt plants in OAM (and OAM Xtra) seed blends. More data are needed to 

estimate larval movement and survival in a 5% and 10% OAM seed blend. 

SWCB data were from one year and two locations, and natural infestation was sparse; only few 

larvae established on refuge plants in seed blends. Hence, it is not surprising that little or no 

movement was observed onto surrounding Bt plants. Dispersal of later instar larvae cannot be 

adequately addressed due to the limited number of SWCB that established on refuge plants. 

Pioneer/DuPont’s preliminary data should be used and interpreted with caution. BPPD 

recommends that additional trials (multi-year, multi-location) be conducted and data be collected 

to better estimate the propensity of larval movement and survival for SWCB. 

Pioneer/DuPont reported that in some cases seed blends caused less susceptible SWCB to 

emerge from refuge plants relative to structured refuges of equivalent non-Bt proportions. BPPD 

believes that a reduction in effective refuge should be expected with a seed blend strategy, 

- 26 - 
especially for a pest that has a great larval mobility (such as observed for ECB and SWCB). The 

key is to determine how great the reduction in effective refuge can be under worst-case 

conditions so that an IRM model can incorporate conservative parameter values in its 

simulations when estimating durability for seed blends. 

Pioneer/DuPont’s efficacy trials showed that OAM seed blends were efficacious against 

lepidoptera stalk boring pests such as ECB and SWCB. No SWCB damage was reported on Bt 

plants (in seed blends) surrounding refuge plants; BPPD notes that this is not surprising 

considering that natural infestation levels were low. Under high infestation levels, damage to 

surrounding Bt plants might have occurred. The applicants concluded from observed damage to 

refuge plants in seed blend plots that blends would serve as functional ECB refuges. BPPD notes 

that refuge plants in seed blend plots may be functional but not equivalent refugia compared to 

structured refugia of similar non-Bt proportions due to a reduction in effective refuge (as 

discussed above). Hence a 5% OAM blend should be expected to produce less susceptible 

insects than a 5% structured refuge. Using Pioneer/DuPont’s worst-case number of 60% 

(reduction in effective refuge for seed blends), BPPD calculated that an actual seed blend may 

have to contain 8.3% non-Bt seed to have a refuge that is equivalent to that of a 5% OAM 

structured block refuge. 

In the applicant’s artificial infestation experiments with CEW and OAM, significantly reduced 

feeding damage was observed on refuge plants in seed blends compared to damage incurred by 

refuge plants with a structured refuge. BPPD concludes that this implies that a reduction in 

effective refuge from a seed blend implementation of OAM can also be expected for CEW. 

BPPD noted that the CEW establishment was lower on refuge plants than on Bt plants and 

ranged from 0.1-0.4 (mean) larvae/ear on non-Bt plants and from 0.1-0.7 (mean) larvae/ear on Bt 

expressing plants (7 out of 10 plants sampled had a larvae. Pioneer reported that there was no 

statistically significant difference between Bt plants in blends and pure stands. Equally important 

though, the numbers of CEW recovered from Bt corn ears was higher than on non-Bt plants; the 

observed ear feeding damage was, however, much lower on Bt than on non-Bt corn. No 

discussion was provided by the applicant to address the results further. BPPD notes that while 

OAM and OAM Xtra may be efficacious against CEW, a significant larval presence on Bt plants 

was still detected. 

Pioneer/DuPont conducted a preliminary efficacy study for OAM and OAM Xtra seed blends 

targeting FAW and was able to demonstrate that both blends showed control of the target pest, 

and at least in OAM blends, leaf damage to refuge plants was significantly reduced compared to 

pure refuge plots. BPPD concludes that for FAW (as well as ECB, SWCB, and CEW) a 

reduction in effective refuge from a deployment of a 5% OAM seed blends should be expected. 

A PIP product expressing both Cry1Ab and Cry1F should be efficacious against susceptible 

FAW. 

B. Modeling 

BPPD recognizes that Pioneer/DuPont conducted an elaborate modeling effort and provided the 

Agency with a transparent description of model structure, assumptions, parameters, and a 

discussion about outputs, sensitivity analyses, and their own interpretations, which facilitated the 

Agency’s review of the applicant’s submission. The following section summarizes and discusses 

- 27 - 
BPPD’s concerns and recommendations with respect to some assumptions and parameter values 

chosen by Pioneer/DuPont and contains recommendations to address important uncertainties. 

Uncertainties and Recommendations: 

a) Model Structure 

Pioneer/DuPont’s model structure was individual based (not frequency based), deterministic, and 

spatially explicit. BPPD agrees with the applicant’s approach of modeling larval dispersal 

explicitly. This allowed Pioneer/DuPont to incorporate dynamics of seed blends that emerge 

from plant-to-plant movement that would otherwise not be captured by any parameter in a model 

that incorporates larval movement implicitly (SAP 2011). Larval movement is discussed further 

in the next section. 

As Pioneer/DuPont pointed out, their simulations were run out past 200 years when local pest 

population densities in the model were below one individual/hectare. This introduced the 

increased importance of genetic drift, which is a stochastic process. Hence, BPPD recommends 

that for simulations where populations are expected to have very low densities that it might be 

beneficial to switch to a stochastic rather than deterministic model. It appears counterintuitive to 

run simulations for so many hypothetical generations when extinction is practically reached in as 

little as 5 generations. However, as long as there are refuges in the landscape that allow for the 

production of susceptible genotypes, the applicant’s approach could be justified. Another 

approach to modeling the durability of OAM could be to artificially inflate the fecundity (many 

fold) of the pest to a point where extinction ceases to occur. Then the model outputs for a 5% 

blend and 5% structured refuge could be compared relative to each other without the interfering 

factor of extinction and neglected consideration of genetic drift. 

b) Assumptions and Parameters 

• Resistance Allele Frequency: The applicant chose an initial Cry 1Ab resistance allele 

frequency of 0.001 (best case) and 0.005 (worst case) for both pests and for Cry1F and 

SWCB. The initial Cry1F resistance allele frequency for ECB was set at 0.01 (best case) and 

0.05 (worst case). BPPD concludes that these assumptions seem reasonable based on 2010 

SAP discussions (SAP 2011). It was not surprising that this key parameter was determined to 

be sensitive in the applicant’s analysis. 

BPPD requests that for future simulations, the applicant report the final resistance allele 

frequency together with the durability estimates. No such FRAF were reported in 

Pioneer/DuPont’s submission to the Agency for this application. 

• Dominance: Pioneer/DuPont stated that they obtained their estimates for ‘h’ by empirically 

testing individuals (SS, RS, and RR) from Cry1F and Cry1A resistant colonies (Pan et al. 

2010, Crespo et al. 2009). For the two toxins and ECB and SWCB, the applicant chose a 

dominance value of 0.01 (best case) and 0.05 (worst-case) and 0.02 (best) and 0.1 (worstcase), respectively, yet incorporated a 2-fold difference in dominance value between 

vegetative and reproductive corn (maximum of 0.1 and 0.2 for ECB and SWCB, respectively 

in worst-case simulations). It was not surprising that this key parameter was determined to be 

sensitive in the applicant’s analysis. 

- 28 - 
With respect to Cry1Ab corn, Crespo et al. (2009) showed that when ECB larvae fed on 

reproductive parts that the dominance value could be much higher than the maximum values 

chosen by Pioneer/DuPont. For example, the dominance values for ‘percentage of survival’, 

‘weight gain’, and ‘larval survival and weight gain’ combined were 0.31, 0.28, and 0.17, 

respectively. The authors further reported that dominance based on LC50 was estimated at ‘h’ 

= 0.27 for reproductive stage plants suggesting that resistance to Cry1Ab was intermediate 

and incompletely recessive compared to other reports in the literature. Survivors were found 

on silk, pollen, ear tips, and kernels in the corresponding plant locations (however, survival 

was measured after 15 days and not followed through to adulthood). Although survival was 

observed on reproductive Cry1Ab corn plants in their greenhouse, no resistant larvae 

survived on the vegetative stage (making resistance functionally recessive for this stage). 

BPPD is aware that lab selected colonies can be selected for minor resistance genes that 

would otherwise not be selected for under field conditions (Crespo et al. reported that 

resistance in their lab selected colony was polygenic and controlled by > five loci). However, 

in light of these data, BPPD recommends that Pioneer/DuPont consider greater dominance 

values for ECB and SWCB worst-case simulations on reproductive stage Cry1Ab corn 

reflective of those reported by Crespo et al. (2009). 

The applicant chose the same dominance values for Cry1F as for Cry1Ab and assumed that 

the resistance allele was recessive. Their selection of parameter values was based on Peirera 

et al. (2008) who demonstrated that resistance in their laboratory selected ECB strain was 

recessive. When RR, RS, and SS-genotypes were exposed to Cry1F expressing corn plants, 

heterozygous resistant individuals exhibited survival rates similar to susceptible individuals; 

however, this similarity was stronger on reproductive maize than vegetative maize (which is 

the opposite of what was reported for Cry1Ab corn). The authors concluded that resistance 

was functionally recessive but more recessive for reproductive than vegetative Cry1F corn. 

Based on percentage of larval survival, percentage of weight gain, and survival and weight 

gain combined, their reported dominance values for vegetative corn were 0.23, 0.27, and 

0.07, respectively. For reproductive corn, those estimated dominance values were 0.10, 0.01, 

and 0.00, respectively. Based on survival and weight, their reported dominance values were 

0.07 and 0.00 on vegetative and reproductive corn. BPPD recommends that for first 

generation ECB and SWCB, which are primarily exposed to vegetative corn that the 

dominance values be increased to 0.07 (using survival and growth combined) for best case 

and 0.23 (using percentage of survival only) for worst case simulations. For dominance 

values on reproductive corn, BPPD recommends increasing worst case assumptions to 0.10. 

Pioneer/DuPont increased the dominance value of the resistance gene by two-fold for older 

instars. BPPD supports this assumption. 

• Survival: Pioneer/DuPont did not report what the actual survivorships of heterozygous 

individuals were for Cry1Ac and Cry1F in the model. This information is important to have 

because heterozygote survival will likely be the driver for resistance evolution (SAP 2011). 

BPPD recommends that the applicant report actual RS-survival data used in the model. 

The applicants reported that susceptible 3rd instar mortality of SWCB (N = 30) was 95.8% on 

1507( x 59122) x MON 810 and 100% on MON 810. In their write-up, the applicants stated 

- 29 - 
that the difference was significant; in their Table 1 (Appendix 2) they stated that the 

difference was not significant. BPPD requests a clarification with respect to the level of 

significance between these results. It needs to be clarified whether there is a potential for 

some level of antagonistic effects between 1507 and MON 810 in SWCB. 

The applicant chose a pre-dispersal tasting survival for ECB and SWCB of 60% and 95%, 

respectively for susceptible genotypes. On the other hand, the dominance values they 

selected in their model were 1% (for ECB) and 2% (for SWCB) for best case and 5% (for 

ECB) and 10% (for SWCB) for worst case. BPPD notes that if pre-dispersal survival is this 

high, that the dominance value should not be chosen to represent a high-dose but rather a low 

dose toxin. Pioneer/DuPont need to revise their modeling analysis to incorporate this 

correction. The applicant’s sensitivity analysis showed that dominance for pre-dispersal 

tasting survival was sensitive, especially at upper values around 0.2 (still too low for a lowdose effect on the target pests); BPPD expects that the durability of OAM seed blends will be 

significantly affected after correcting the value for this parameter to reflect realistic survival 

of instars. 

• Larval Movement: Pioneer/DuPont included one pathway of mortality from toxin exposure 

that was mentioned by the SAP (2011), namely the pre-dispersal tasting mortality. Inclusion 

of this parameter addresses the BNI movement hypothesis (SAP 2011). However, the 

applicant’s model lacks inclusion of the second toxin mortality pathway associated with 

movement: the NBI hypothesis addresses mortality associated with RS genotypes that 

originate and remain during most of their development on non-Bt but move as late-instars 

onto Bt where they have a certain probability of survival. BPPD recommends that 

Pioneer/DuPont incorporate this particular larval movement scenario/mortality for ECB and 

SWCB into their model. BPPD recognizes that there are uncertainties with respect to 

heterozygous larval movement from non-Bt to Bt and subsequent survival rates on single PIP 

and pyramided PIP; in light of these uncertainties, Pioneer/DuPont should choose 

conservative values for this parameter. 

Specifically, Pioneer/DuPont modeled ECB and SWCB first instar movement from non-Bt to 

Bt with a benchmark value of 0.76. Onstad and Gould (1998) have shown that the probability 

of ECB larvae leaving a refuge plant is approximately 90% and the probability of leaving a 

Bt plant was 98%. The applicants incorporated Onstad and Gould’s probability for leaving a 

Bt plant but should have also included the probability for larvae leaving refuge plant. In 

addition, the probability of leaving a refuge plant should be set higher for species that are 

cannibalistic, such as SWCB (SAP 2011). If the applicants had incorporated such a 

benchmark value for dispersal from non-Bt to Bt plants, the estimated durability for the seed 

blends may likely be lower than predicted. Greater larval (inter-plant) dispersal in a block 

refuge should not affect the estimated final durability. 

• Movement of Adults: Pioneer/DuPont assumed that 25% of the mated SWCB females 

dispersed and laid their eggs within 800 meters of the location that they mated. For ECB, it 

was assumed that 100% of the females dispersed and oviposited in such a manner. BPPD 

notes that there is some uncertainty with respect to the amount of SWCB females that leave 

their natal fields. However, Rolston (1955) and Qureshi et al. (2006) found that female 

SWCB disperse and oviposit much like ECB. BPPD recommends that Pioneer/DuPont 

- 30 - 
increase the percentage of dispersal for pre-ovipositional SWCB females to address this 

uncertainty. 

Langille and Keaster (1973) observed that males engaged in a preferred pre-copulatory flight 

behavior within their natal fields. Hence, BPPD concurs with Pioneer/DuPont’s assumption 

that SWCB males mate primarily in their natal fields. 

• Carrying capacity: the model flow chart provided in MRID 480707-01 (Figure 1, p.10) 

outlined that after toxin mortality and plant-to-plant dispersal occurred in the applicant’s 

model that the carrying capacity was applied to larval densities. “Larvae in excess of the 

carrying capacity of the plant [did] not survive”. This appears to be one standard approach to 

address density-dependence, although it may not be the best (M. Caprio 2011, personal 

communication) 

• Uniform egg dispersal: The applicant assumed uniform oviposition (egg dispersal) of ECB in 

seed blends and stated that a large body of evidence suggested that Bt protein in maize did 

not affect ECB ovipositional behavior (Hellmich et al. 1999). BPPD agrees with 

Pioneer/DuPont that this assumption may generally be accurate recognizes that with a seed 

blend strategy (i) plants may incur root damage from CRW, which slows plant grow and can 

cause crowding out of non-Bt plans by faster growing Bt plants and (ii) second generation 

ECB can detect damaged refuge plants from non-damaged Bt plants (SAP 2011) and respond 

by ovipositing onto non-damaged (Bt) plants with greater likelihood. Crowding out and 

detection of damaged refuge plants in a blended environment can be mechanisms which lead 

to greater non-uniform oviposition and greater oviposition onto Bt plants. This ability to 

discriminate between refuge and Bt plants is not a concern for structured refuge strategies. 

(SAP 2011) 

BPPD recommends that Pioneer/DuPont include a degree of non-random oviposition for first 

generation as well as second generation ECB and SWCB where eggs will be laid with greater 

probability onto Bt plants in seed mixes than on non-Bt plants. 

• Epistasis: Epistasis refers to the interaction of genotypes at different loci and is usually 

defined as the suppression of a gene at one locus by a gene at another locus. The SAP (2011) 

recommended that different mechanisms of epistasis be considered for products expressing 

pyramids of two or more toxins. As was discussed by the SAP, there are five heterozygous 

genotype combinations in a two locus case (as is applicable for OAM), and any change in 

their survival values could have differing impacts on the evolution of resistance (SAP 2011). 

However, even if multiplicative survival for heterozygotes in a two toxin case was assumed 

(no epistasis), the SAP cautioned that “the impacts of multiple toxins [in a pyramid] on the 

rate of resistance evolution may not combine in a simple way” and the rate of evolution at all 

loci could be determined by the fastest evolving locus. 

 

The applicant included a low-level cross resistance between Cry1A and Cry1F (CR1Æ2) for 

their best and worst-case scenarios, which was determined to be sensitive in the applicant’s 

analyses. Cross-resistance is one form of epistasis that the SAP recommended including for 

those particular Cry-toxins. BPPD recommends that a low level of cross resistance between 

Cry1F and Cry1A (CR2Æ1) for a best-case (not only worst-case) scenario modeled be also 

included. Pioneer/DuPont’s studies showed that there were some shared binding sites for the 

- 31 - 
two toxins in ECB, although multiple ligand blotting supported that each toxin also had 

multiple unique binding sites (BPPD 2010c). 

BPPD recommends that the applicant include a mechanism for possible developmentally 

restricted expression of low levels of Cry-protease where older ECB and SWCB larvae 

survive Bt exposure when moving from non-Bt onto Bt. The SAP mentioned that this may be 

a more likely mechanism for these two target pests (SAP 2011). 

c) Sensitivity Analysis 

Pioneer/DuPont conducted sensitivity analyses for parameters of concern by changing one 

variable at the time over a predetermined range. BPPD notes that if the applicant incorporated a 

probability analysis for parameters of uncertainty into their model that a statistical approach 

could be used to determine which parameters are significantly sensitive and/or have the greatest 

effect on the results (i.e. step-wise regression, standard regression, Bayesian model analysis, etc). 

In addition, a probability analysis would also allow the registrant to provide a risk level profile 

(0%, 5%, and 50%) for their simulation results. For the purpose of relative comparisons between 

different refuge strategies rather than a comparison of absolute values, it would be helpful to the 

Agency to have such a risk profile. 

Pioneer/DuPont tested a multitude of parameters for their sensitivity to changes in value (see 

Table 4). Among other things, they explored the impact of increased susceptible late-instar larval 

survival to the toxins expressed in the pyramid (OAM) and reported that no sensitivity was 

apparent. BPPD notes that it would be more relevant to explore the sensitivity of heterozygote 

survival (maybe late-instar) since this presents an uncertainty for both ECB and SWCB per se 

and drives resistance evolution in seed blends when coupled with interplant movement (Mallet & 

Porter 1992). The SAP (2011) recommended exploring survival of RS-individuals that is in 

excess of 5x the survival for SS-individuals. 

d) Pioneer/DuPont Results 

The applicant estimated that for ECB, the durability of OAM would last more than 200 years 

under best-case assumptions with a seed blend or structured refuge strategy. BPPD concludes 

that it is insufficient to report “greater than”-results because actual numbers of predicted years of 

durability should not be taken literally. Rather, it is the relative durability between a 5% seed 

blend and a 5% structured refuge for OAM that should be assessed. BPPD needs to determine 

what the relative difference in durability (based on the SAP 2011 report) and risk of resistance 

evolution would be by switching from a structured refuge to a seed blend approach. A two toxin 

blend with a 5% seed blend may be more durable than a single toxin with a 20% block refuge, 

but BPPD is unable to quantify the durability because no comparative modeling analysis was 

provided by the applicant. 

When the applicant used worst-case modeling assumptions for ECB, the estimated durability for 

a structured refuge and seed blend was 35 years and 26 years, respectively. This represents 

approximately a 1.4 fold decrease of in durability for the seed blend. It is unclear whether this 

difference was statistically significant or whether the applicant was in a position (based on their 

model approach) to use statistical tools to determine the level of significance for the difference 

between the two results. BPPD recommends that the applicant use a probability approach to 

- 32 - 
modeling parameters of uncertainty, which would make it possible to determine 1) whether the 

estimated durability between blend and block refuge differed significantly and 2) which 

parameters were most important/sensitive in the model. 

The applicant estimated that for SWCB, the durability of OAM would last more than 200 years 

under best-case assumptions with a seed blend or structured refuge strategy. As stated above, it is 

insufficient to report “greater than”-results because actual numbers of predicted durability should 

not be taken at face value and a relative comparison between actual “durability years” may be 

more meaningful. When Pioneer/DuPont used worst-case parameter assumptions, the estimated 

durability for blocks was still >200 years when the refuge was not moved, yet it decreased to 11 

years when the refuge was moved yearly. The seed blend durability was estimated at 16 years 

(slightly more than the block with refuges moved). This represents approximately a 12.5 fold 

decrease in durability by switching from a block to a seed blend strategy under worst case 

assumptions using the applicant’s model construct. BPPD reiterates the SAP’s recommendation 

that an IRM strategy needs to be designed around the target pest that is at greatest risk of 

evolving resistance and for which most uncertainties are identified: in this case, the pest would 

be SWCB. 

Extinction was reached in the seed blend and structured refuge simulations after three years for 

ECB and four (and five) years for SWCB. The applicant discussed the importance of genetic 

drift at this point and also added that results beyond this point should be considered with caution. 

In another part of their submission, Pioneer/DuPont contended that while it was a key 

consideration whether refuge plants in a Bt seed blend produced sufficient amounts of 

susceptible insects to effectively delay resistance evolution, the number of insects emerging from 

refuge plants in OAM seed blends were sufficient to provide a durability of >200 years. BPPD 

notes that these two statements contradict each other and that the second statement is not 

supported by the rapid extinction events reported by the applicant. BPPD agrees, however, with 

the first point made that low population densities increase the importance of considering genetic 

drift. Earlier in this review BPPD recommended that Pioneer/DuPont increase the fertility of the 

target pests artificially to a point where population extinction does not occur any longer. Final 

estimated durabilities for blends and structured refuges could then be compared relative to each 

other. In this manner, Pioneer/DuPont could avoid having to deal with genetic drift and 

incorporating a stochastic process into their model structure. 

e) The SAP’s (2011) two-gene analysis for a 5% seed blend and 5% block refuge using 

Ives et al. (2011) 

The SAP (2011) conducted an extensive theoretical analysis using Ives et al.’s model (in press), 

which was described in the SAP report submitted to the Agency (and publicly available). This 

was done by comparing the estimated durability of a structured refuge compared to that of a 5% 

seed blend under different assumptions (i.e. heterozygote survival, dose, epistasis, adult and 

larval movement). The SAP demonstrated that assuming moderate lepidopteran larval movement 

and density-dependence, effectively no seed blend percentage provided durability equivalent to 

that of the structured refuge with equivalent non-Bt refuge. The results still represented an 

overestimate of seed blend durability because the Panel did not incorporate selection in the corn 

ears and explore larval movement and epistasis sufficiently. The Panel stated that in addition to 

seed blends (with high-dose toxins causing low RS-survival) being less durable than structured 

- 33 - 
refuges with the same non-Bt percentage, seed blends had also greater uncertainties associated 

with them than structured refuges due to lack of information about ECB and SWCB larval 

movement and how such movement affected heterozygote survival. The SAP concluded that 

based on the uncertainties and their modeling analysis that at the moment, a seed blend for 

lepidoptera was not scientifically supported. BPPD notes that the SAP (2011) specifically looked 

at a 5% SmartStax seed blend (a separate Bt corn product), but their conclusions are applicable 

to other Bt corn seed blends with two-toxin pyramids. 

f) BPPD overall conclusion 

Pioneer/DuPont did not report the actual numbers of generation to theoretical product failure, 

and using the reported results from the applicant’s model construct, the Agency is unable to 

determine what the relative loss in durability would be for a seed blend with two toxins 

compared to the presently approved 5% structured refuge. All models (whether complex or 

simple) are idealizations and abstractions of reality, have incorporated those mechanisms and 

assumptions that the model designers feel are important, and contain an inherent amount of 

uncertainties. Parameters that are used in the model are at best ‘good estimates’ and in some 

cases ‘best guesses’ and do not represent actual values encountered in nature. The SAP (2011) 

therefore recommended that scientists and foremost regulatory agencies should pay more 

attention to relative comparisons between various model outputs rather than their absolute 

values. BPPD requests that Pioneer/DuPont report final estimated number of years of durability 

including actual final resistance allele frequencies for their model scenarios so that a percent 

difference can be determined between various strategies and make the recommended changes to 

their model as discussed throughout the review (and summarized below). 

Although Pioneer/DuPont applied for a 5% OAM and 10% OAM Xtra seed blend, BPPD 

focused its analysis of the applicant’s submission on the 5% OAM seed blend because all of the 

modeling was done for a 5% blend. The applicant should submit a modeling analysis for a 10% 

OAM Xtra seed blend targeting SWCB and ECB. In light of the uncertainties with the 5% seed 

blend proposal, such an additional modeling analysis would allow the Agency to assess the 

durability of a greater blend percentage. Generally, BPPD would expect a 10% seed blend to be 

more conservative and more durable than a respective 5% seed blend with two lepidoptera 

toxins. Carriere et al. (2004) concluded that differences in Bt durability predicted by block and 

seed blends (caused by a reduction in refuge insects in seed blends) could be overcome by 

increasing the percent of non-Bt plants in seed mixtures. However, BPPD cannot quantify the 

durability without a comparative modeling analysis. BPPD also recommends that the applicant 

model the durability for a single gene PIP with a 20% structured refuge so that a relative 

comparison between the estimated durability of all four strategies (single PIP block, 5% and 10% 

seed blend w/pyramid, and 5% block with pyramid) can be made. 

The SAP (2011) demonstrated that a dual gene, high efficacy product with a seed blend approach 

targeting ECB and SWCB was less durable than a block refuge of equivalent non-Bt proportion. 

It was unclear however from their report what the relative loss in durability would be for a seed 

blend under Ives et al.’s (2011) model construct. 

The SAP predicted that SWCB might be the species at greatest risk of evolving resistance and 

hence an IRM strategy should be designed around this pest. Under worst-case model 

assumptions and using Pioneer/DuPont’s model construct, there was a 12.5 fold loss in durability 

for OAM targeting SWCB when the IRM strategy was switched from a 5% structured refuge to a 

- 34 - 
5% seed blend. It is likely that a 5% structured refuge for OAM is a more conservative IRM 

approach for SWCB (as well as ECB). 

There are various uncertainties and deficiencies with Pioneer/DuPont’s model submission for 

OAM, and BPPD is unable to confidently assess the risk of resistance evolution for a 5% OAM 

seed blend targeting ECB and SWCB due to these uncertainties. BPPD recommends that 

Pioneer/DuPont conduct additional modeling to address and incorporate the following 

recommendations, uncertainties, and deficiencies: 

• Report final resistance allele frequency and actual estimates of durability rather than 

‘greater than’ because relative comparisons may be meaningful even if actual numbers 

are not. 

• Artificially inflate fecundity of the pest to 1) avoid extinction, 2) introduce importance of 

genetic drift, and 3) determine relative durability for a seed blend and structured refuge. 

• Incorporate a probability analysis to address uncertainties with various important 

parameter values and to provide the Agency with a risk profile (i.e., 0%, 5%, and 50%) 

for OAM and the modeled IRM strategies as well as a level of significance between 

estimated durability for different strategies. 

• Provide comparative modeling (incorporating BPPD’s recommendations in this review) 

to evaluate: 

- 20% block refuge (single toxin) 

- 5% block refuge (pyramid) 

- 5% blended refuge (pyramid) 

- 10% blended refuge (pyramid) 

• Correct the dominance value for pre-dispersal mortality/survival to be reflective of a lowdose exposure rather than a high-dose exposure. 

• Include non-uniform oviposition parameter for first generation lepidoptera in OAM Xtra 

and second generation adults in OAM seed blends. 

• Include NBI-movement hypothesis to address mortality associated with RS genotypes 

that originate and remain during most of their development on non-Bt but move as lateinstars onto Bt where they have a certain probability of survival. 

• Focus IRM strategy on species with greatest uncertainties and risk of evolving resistance 

– in this case southwestern corn borer. 

• Research on adult movement (related to mating and movement from refuges), larval 

movement, larval feeding (i.e., selective feeding within corn ears or on pollen), survival 

of heterozygote genotypes on OAM (markers may need to be determined for 

heterozygotes), and the potential for epistatic mechanisms of resistance (particularly with 

older instars). 

 

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V. REFERENCES 

MA Caprio. 2011. Personal communication with JC Martinez, US EPA/OPP/BPPD. 

Carriere Y, MS Sisterson, and BE Tabashnik. “Resistance management for sustainable use of 

Bacillus thuringiensis crops in integrated pest management”, p. 65-95. Book chapter in “Insect 

Pest Management: Field and Protected Crops”, Springer, 2004. 

Crespo ALB, TA Spencer, AP Alves, RL Hellmich, EE Blankenship, LC Magalhaes, and BD 

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.

www.interscience.wiley.com, DOI 10.1002/ps.1793 

Davis PM and DW Onstad. 2000. Seed mixtures as a resistance management strategy for 

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protein. Journal of Economic Entomology, Vol. 93 (3): 937-948 

Dorhout DL, TW Sappington, and ME Rice. 2008. Evidence for obligate migratory flight 

behavior in young European corn borer (Lepdioptera: Crambidae) females. Environmental 

Entomology, Vol. 37: 1280-1290 

Dorhout DL, TW Sappington, and ME Rice. 2011. Flight behaviour of European corn borer 

infected Nosema pyrausta. Journal of Applied Entomology, Vol.135: 25-37 

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European corn borer in eight inbred lines of dent corn. Research Bulletin 860. Ohio Agricultural 

Experimental Station, Wooster, Ohio. 

Hellmich RL, LS Higgins, JF Witkowski, JE Campbell, and LC Lewis. 1999. Oviposition by 

European corn borer (Lepidoptera: Crambidae) in response to various transgenic corn events. J. 

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structured environments. Ecology Letters, Vol. 5: 792-801 

Ives AR, PR Glaum, NL Ziebarth, and DA Andow. 2011. The evolution of resistance to two 

toxin pyramid transgenic crops. Ecol. Appl. in press. 

Knutson AE and FE Gilstrap. 1990. Population dynamics and natural enemies of the 

southwestern corn borer (Lepidoptera: Pyralidae) in the Texas High Plains. Ph.D. Dissertation, 

Texas A&M University, College Station, TX. 

Mallet J and P Porter. 1992. Preventing insect adaptation to insect-resistant crops: are seed 

mixtures or refugia the best strategy? Proceedings of the Royal Society, London B, 250: 165-169 

Onstad DW and F Gould. 1998. Modeling the dynamics of adaptation to transgenic maize by 

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593 

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Onstad DW, PD Mitchell, TM Hurley, JG Lundgren, RP Porter, CH Krupke, JL Spencer, CD 

DiFonzo, TS Baute, RL Hellmich, LL Buschman, WD Hutchson, and JF Tooker. Seeds of 

change: corn seed mixtures for resistance management and IPM. Journal of Economic 

Entomology, Vol.104(2):343-352 

Pan Z, A Alves, Q Chen, JL Flexner, B Stanley T Rood, and J Zhao. 2010. Predictive model 

supporting reduced block/strip lepidopteran refuge for 1507 x MON810 maize in the Corn Belt. 

Pioneer Hi-Bred International, Inc. Internal study report (MRID48005602) submitted to the 

Agency. 

Porter P. 2011. Email communication with JC Martinez, US EPA/OPP/BPPD. 

Qureshi JA, LL Buschman, JE Throne, and SB Ramaswami. 2006. Dispersal of adult Diatrea 

grandiosella (Lepdioptera: Crambidae) and its implications for corn borer resistance management 

in Bacillus thuringiensis Maize. Annals of the Entomological Society of America, Vol. 99 (2): 

279-291 

JE Rolston. 1955. The southwestern corn borer in Arkansas. Ark. Agric. Exp. Stn. Bull. 

Fayetteville 553. 

Ross SE and KR Ostlie, 1990. Dispersal and survival of early instars of European corn 

borer (Lepidoptera: Pyralidae) in field corn. Journal of Economic Entomology, Vol. 83 (3): 831-

836. 

BE Tabashink. 1994. Delaying insect adaptation to transgenic plants: seed mixtures and refugia 

reconsidered. Proceedings of the Royal Society, London B, 255: 7-12 

White JA and DA Andow. 2007. Foraging for intermittently refuged prey: theory and field 

observation of a parasitoid. Journal of Animal Ecology, Vol. 76:1244-1254 

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Other: 

EPA Scientific Advisory Panel. 1998. Transmittal of the Final Report of the FIFRA Scientific 

Advisory Panel Subpanel on Bacillus thuringiensis (Bt) plant-pesticides and resistance 

management, Meeting held on February 9 and 10, 1998 

EPA Scientific Advisory Panel. 2000. Transmittal of meeting minutes of the FIFRA Scientific 

Advisory Panel meeting held October 18-20, 2000 on Bt plant-pesticides risk and benefit 

assessments: insect resistance management. Report from P. Lewis (DFO) dated March 12, 2001. 

EPA Scientific Advisory Panel. 2009. Transmittal of meeting minutes of the FIFRA Scientific 

Advisory Panel meeting held February 23-24, 2009 on the evaluation of the resistance risks from 

using a seed mix refuge with Pioneer’s Optimum AcreMax1 corn rootworm-protected corn. 

Memorandum from J.E. Bailey (DFO) to Dr. D. Edwards (Director of OPP) on May 6, 2009. 

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- 38 - 

EPA Scientific Advisory Panel. 2011. Transmittal Meeting Minutes of the FIFRA Scientific 

Advisory Panel Meeting Held December 8-9, 2010 to Address Scientific Issues Associated with 

Insect Resistance Management for SmartStaxTM Refuge-in-the-Bag, a Plant-Incorporated 

Protectant (PIP) Corn Seed Blend. Memorandum from Dr. Sharlene Matten (DFO) to Dr. Steven 

Bradbury (Director of OPP) on March 3, 2011. 

BPPD, 2008a. Review of studies submitted by Pioneer in support of a New Use Sec(3) product 

registration for Optimum AcreMax1 Insect Protection, also known as Herculex 

XTRA(Cry34/35+Cry1F, EPA Reg. 29964-A) and Herculex I (Cry1F). Memorandum from 

J.C.Martinez to M. Medelsohn on December 17, 2008. 

BPPD. 2008b. Preliminary Review of the Modeling Chapter and Trait Durability Plan Submitted 

by Pioneer in Support of a New Use Sec(3) Product Registration for Optimum® AcreMaxTM 1 

Insect Protection, also known as Herculex XTRA. Memo from J.C. Martinez to M. Mendelsohn 

dated December 17, 2008. 

BPPD. 2010a. Science Assessment of Pioneer’s modeling addenda (MRIDs 478710-01, 478879-

01, and 479108-01) in support of their application of registration for Optimum AcreMax1 Insect 

Protection. Memo from J.C. Martinez to M. Mendelsohn dated February 4, 2010. 

BPPD. 2010b. Science risk assessment to evaluate the durability of a 10% Cry34/35 seed blend 

refuge deployment of Cry34/35 corn relative to the durability predicted for a 20% block in 

regards to Pioneer/DuPont’s Optimum AcreMax1. Memo from J.C. Martinez to M. Mendelsohn 

dated April 22, 2010. 

BPPD. 2010c. Review of insect resistance management (IRM) considerations with a proposed 

reduced refuge for Bt corn events 1507 x MON 810 and 1507 x 59122 x MON 810 in cotton 

growing areas. Memo from A. Reynolds to A. Sibold dated October 20, 2010.