CONCLUSIONS AND RECOMMENDATIONS
BPPD has reviewed Pioneer/DuPont’s applicat ion for a 5% Optimum AcreMax seed blend (Cry1Ab and Cry1F) and 10% Optimum AcreMa x Xtra seed blend (Cry1Ab, Cry1F, and Cry34/35) targeting lepidoptera pest of corn (E CB and SWCB) and has concluded that there are a number of concerns and uncertainties with th e 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 requi rements (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 pest s exceeded 200 years when benchmark parameter assumptions were used, while they estimated th at population extinction c ould be reached in less than 10 years. BPPD concluded that the importan ce of genetic drift should not be ignored when populations are below one individua l/hectare and that the Pioneer /DuPont’s durability estimates are therefore uncertain. BPPD made recommendati ons on how to address this uncertainty. Under the applicant’s worst-case model assumptions (act ual durability estimates were provided in this case and given in years to resistance) and Pi oneer/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 st rategies should be designed around the pest complex at greatest risk of e volving resistance. BPPD conclude s that for OAM, the main focus may likely have to be on an IRM strategy for SW CB. BPPD also concluded that some parameter values needed to be more conservative (i.e., cr oss resistance, several a ssumptions for dominance) and other important aspects of larval movement and surv ival 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 toxi ns 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 amou nt of uncertainties. The SAP (2011) therefore recommended that more attention should be paid to relative comparisons between various model outputs rather than thei r 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 Cry34/35A b1 (aimed at control ling both lepidoptera and corn rootworm pests of corn). In light of the uncertainties wi th the 5% seed blend proposal mentioned throughout this review, such an additi onal modeling analysis would have allowed the Agency to assess the durability of a greater bl end percentage. Generall y, 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 si ngle toxin seed blend strategy for CRW (5% and 10%), and BPPD conclusions are containe d 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 incorp orate 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 comparis ons between various strategies may be meaningful even if act ual numbers are not..

• Artificially inflate fecundity of the pest to 1) avoid extinction, 2) introduce importance of genetic drift, and 3) determine relative durabil ity for a seed blend and structured refuge.

• Incorporate a probability anal ysis to address uncertainti es 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 mo rtality/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 a ssociated 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 grante d FIFRA section 3 registratio ns on February 24, 2010. The registrations are for pyramided products contai ning the Cry1F (event 1507) and Cry1Ab (event MON 810) toxins, which were derived by conventi onal breeding from Pion eer’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 cont rol of corn rootworm.] Both Cry1F and Cry1Ab are active against lepidopteran stalk bori ng pests, including European corn borer (ECB, Ostrinia nubilalis ), corn earworm (CEW, Helicoverpa zea ), and southwestern corn borer (SWCB, Diatraea grandiosella ). A number of other secondar y lepidopteran pests including fall armyworm (FAW, Spodoptera frugiperda ) and western bean cutworm (WBCW, Richia albicosta ) are also controlled or suppressed by Cr y1F and/or Cry1Ab. The registration was granted with a 20% refuge in the Corn Belt a nd 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 th e 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 AcreMa x Xtra, OAM Xtra). Supporting data for this request are contained in MRIDs 480707-01, 480707-02, and 481262-01.
Optimum AcreMax1 Insect Prot ection (OAM1) is a precursor to the requested Optimum AcreMax Xtra registration. In February, 2008, Pi oneer Hi-Bred International, Inc. submitted the original application to regist er OAM1, which is a corn seed blend product that contains two different kinds of seeds from the already regist ered 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 ref uge 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 Scie ntific 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 fo r 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 literatu re. However, the Panel agreed that a seed mix strategy would lend it self 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 He rculex 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 asse ssments in collaboration with EPA/ORD (BPPD 2010a, 2010b), in which the BPPD IRM Team conc luded 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 porti on 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, Fungi cide and Rodenticide Act (FIF RA) requires that registered pesticide uses do not pose unreasonable adverse e ffect to the environment. The purpose of this risk assessment is to provide insight into the pot ential effects to the environment as measured by the risk of resistance evolution in Bt corn target pests (lepidopte ra) 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 cu rrent OAM registration by replacing the 5% structured refuge for lepidopt era with a 5% seed mixture and applying for a new stacked registration for OAM Xtra deploy ed as a 10% seed blend in the Corn Belt only. This risk assessment focuses on assessing the risk of resist ance 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 Agen cy 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 Pi oneer’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 cr op seeds have been a topic of discussion for approximately two decades. While such an Inse ct Resistance Management (IRM) strategy has obvious benefits ( i.e., no grower compliance component, f acilitating the plan ting of Bt and refuge fields), a variety of re searchers have indicated that under some circumstances this approach could lead to more ra pid evolution of resistance in so me target pests (e.g., Mallet and Porter, 1992; Onstad and Gould, 1998; Da vis and Onstad, 2000; and others).
Scientific Advisory Panels (1998 and 2000) discour aged the Agency from the use of Bt seed mixtures to control lepidopter a target pest because substan tial 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 PI Ps deployed in seed blends for lepidoptera because of remaining uncertainties with larval movement and survival, especially for heterozygous genotypes (RS), and va rious unexplored epistatic eff ects. However, both the SAPs (2009 and 2011) determined that the seed ble nd strategy targeting corn rootworm would lend itself to the pest’s biology.
Theoretical work by Mallet and Porter (1992) show ed that insect resistance was accelerated in seed mixtures compared to Bt stands where 10% of the population was no t 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 (avoi dance of toxic substances), then effective dominance was increased, and seed mixtures could delay resistan ce evolution but that predicted outcomes depended also on other circumstances.
Using Mallet and Porter’s model, Tabashnik (19 94) reported that 10% Bt seed mixtures were more durable than pure Bt stands and that bloc k 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 increase d from 0-50%, Tabashnik fu rther reported that a block refuge strategy performed equally well or better than seed mixtures. However, seed mixtures in conjunction with re fuges were the most durable of all deployment strategies evaluated.
Onstad and Gould (1998) recommen ded 20% block refuges adjacent to the Bt fields over seed blend strategies because of un certainties surrounding expression of high-dose against the target pest. Their modeling results predicted that bloc k refuges would be more durable than seed mixtures aimed at contro lling lepidopteran pests.
In a 2-yr field study with seed mixtures expr essing 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 di spersed 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 pl ants onto Bt plants wo uld result in a reduced effective refuge. They reported that neonate di spersal away from Bt plants was greater than dispersal observed from non-Bt plan ts and noted that extensive use of seed mixtures could select for populations with improved dispersal capab ilities. 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 Taba shnik (1994) were caused by a reduction in refuge insects in seed mixtures that had moved from non-Bt pl ants to Bt plants and could be overcome if the percent of non-Bt plants in seed mixtur es was increased (compared to blocks).
Onstad et al. (2011) discussed di fferent 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 appr oved 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 plan ts, arrive on Bt plants, and are subsequently killed, then seed mixtures can be expected to reduce the refuge po pulation to less than what can be expected to emerge from a block refuge. In either case, literature sugges ts that such conditions would increase the risk of resistance e volution 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 4 th and 5 th 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 expr essed 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 hetero zygous 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 includi ng assumptions of epistasis in the simulation model, and they specifically listed three types of mechanisms by which epistasis could manifest itself and resistance evolution coul d be sped up: a) low-level e xpression of Cry-proteases (more likely the case in species such as Helicoverpa zea ), b) developmentally restricted expression of low levels of Cry-proteases (w hich may apply for late-instar ECB and SWCB), and c) crossresistance.

3. Refuge Effectiveness in Seed Blends ECB adults do not appear to det ect the difference between Bt and non-Bt plants; however there is evidence that adult females disc riminate between plants with and without damage. Additionally, in maize fields planted using a seed blend stra tegy, non-Bt plants can incur root damage from corn rootworm, which slows dow n plant growth. In such inst ances, 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 n on-Bt plants is a concern for seed blends rather than for block refuges. For first generation E CB laying their egg masses in a seed blend field expressing CRW PIPs, ovipos ition may be non-uniform. If there are no CRW traits expressed in the seed blend or no CRW infestations in the co rn fields, the assumption that first generation ECB oviposit egg masses uniformly may be appropr iate. 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 neona te 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 st rategy with equal proportio n 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% bloc k refuge. Larval movement off ref uge plants and decreased larval movement from surrounding Bt plants onto non-Bt pl ants may lead to a reduction of adults in seed blends compared to block refuges of e quivalent 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 regi ons, 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, c ontribute 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 acceler ate resistance in ear feeding Lepidoptera including corn earworm and fall armywo rm. For a 5% seed blend, 95% of the plants will be Bt and 5% will be non-Bt pl ants; 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 enc ountered 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 sec ond 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 anothe r kernel. This ability to move to another source of kernel in this mosaic of toxins and non-toxin environment will giveheterozygous individuals a great fitness advantage; the function al dominance of the resistance allele will increase. None of the refuge plants in the seed blend fi eld will be true-refuge plants to kernel feeders. (Porter 2 011, 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 evolut ion 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 greates t 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 pest s and presents the applicant’s re sults 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 br eeding pyramid of Cry1F a nd Cry1Ab (and stack with Cry34/35Ab1) or 100% refuge plot s. The experimental set up wa s a randomized complete block design with 3 blocks. Artificial infestations we re 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 samp led (representing the pure stand portion of the blend) and investigated for surv iving larvae. In the pure stand re fuge plot, three non-consecutive plants were sampled, and numbers of live larvae were determined. A repeated measure analysis of variance wa s conducted using the s quare-root transformed averages and using the correspondin g number of plants as weights. The main effect of treatment was analyzed as a randomized complete block st ructure with a random block effect. The main effect for plant type and the inte raction 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 gene ration ECB did not establish well at any of the five locations based on the low recovery rate from refuge plants in all treatments (see Ta ble 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 mo derate and variable and ranged from 1.68 - 8.14 larvae/plant in pure stand refuge plots and from 1.31 - 3.51 larvae/pl ant in seed blend plots. The recovery of larvae was significantly greater in pu re 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 gene ration ECB on Bt plants in pure stands and seed blend plots (immediately adjacent or near refuge plant) was negligible and no significant differencesbetween Bt treatments and distance away from ref uge plants were noted (Table 2). The highest recovery rates for second generation ECB were obs erved 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 moveme nt from refuge to protected Bt plants which expressed a high dose pyramid.

Table 2 Mean number of first and second ge neration 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 rate s 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 ge neration ECB (range 0.0- 0.13). Second generation establishment on refuge plants ra nged from 1.31-3.35 larvae/plants.

Pioneer/DuPont Conclusions:
“Data from this study show that deployme nt of 1507 x 59122 x MON810 maize with blended refuge as low as 5% can produ ce susceptible ECB at rates comparable to pure stand refuge. However, insect production from blended ref uge plants was significan tly less than insect production from pure stand of refuge 50% of th e 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 bot h 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 locatio n each in Kansas and Tennessee in 2009 to determine SWCB larval survival from neonate to la te instar when exposed to different seed blend percentages (5% and 10%) expr essing a breeding stack of Cr y1F and Cry1Ab (as well as Cry34/35Ab1) or 100% refuge plot s. 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 rate s 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 0.0 1-0.03 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 diffe rences between treatments were noted.
Survival on refuge plants and immediately adjace nt Bt plants in 5% and 10% seed blends was significantly different between tr eatments at both locations. At Garden City (KS), for example, 0.01 larvae/refuge plant in 5% seed blend plots and no survivors on adjace nt Bt plants were noted. Survival in refuge plots wa s significantly higher than on Bt pl ants (distant to refuge plant) in seed blends.

Pioneer/DuPont Conclusions:
“Data from this study show that deployme nt of 1507 x 59122 x MON810 maize with blended refuge as low as 5% can produ ce susceptible SWCB. The results suggest that blended refuge plants do serve as a functional SWCB refuge, a lthough at one location bl ended 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 Wi ndfall (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 MON 810 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 1 st

and 2 nd generation ECB. Injury from first generati on ECB leaf feeding was recorded using a 9 to 1 visual rating scale where a score of 9 indica ted ‘no injury’ and a score of 1 indicated ‘heavy injury’. The visual rating scal e 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 centim eters. The experimental unit for this study consisted of a plot of maize eight rows wide and 5 2.5 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 e ffects and Fisher’s protected LSD test was used to determine differences among least squares mean s when appropriate. Three separate analyses were conducted on the efficacy data: comparison betw een 1) ECB injury to refuge plants in 5% blends and refuge plants in pure stand is oline 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 ne xt 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 plot s (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, th ese observed differences between treatments were statisti cally significant (data tables no t shown but were provided in MRID 480707-02). When the efficacy was compared between Bt plan ts 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 firs t 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). Sim ilarly, 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 remove d from refuge plant) from seed blend plots (data tables not shown he re but were provide d in MRID 480707-02).

Pioneer/DuPont Conclusions: “The 1507 x 59122 x MON810 maize plants in close pr oximity 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 st and plantings. Some damage observed to refuge plants in seed blends indicates, however, that re fuge 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 mai ze 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 th e experimental design and statisti cal 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 dete rmined by splitting the lo wer eight internodes of each corn stalk sampled and recording the total amount of tunneling per plant in centimeters.

Results: No statistically significant dama ge was observed between refuge plants in seed blends and pure stand plots. Damage from stal k tunneling in seed blends ra nged from 2.4-5.1 cm and from 3.6- 6.2 cm in 100% refuge treatments. No damage wa s observed on Bt plants in pure stands and seed blends (adjacent to and two plants removed fr om 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 pr oximity 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 diffe rent 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 neona tes /plant at the first infesta tion and 25 neonates/plant at the second infestation date (except at Johnston, where larvae from firs t 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 wh ere “9” indicates “no damage” and “1” indicates “heavy injury”. The expe rimental unit in this e xperiment consisted of a block that was four rows wide and 17.5 ft long. E fficacy of all treatments was assessed in a five plant cluster in rows 3 and 4.

- 15 -Analysis of variance was used to analyze main e ffects 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 da mage from FAW was detect ed on refuge plants in 5% OAM Xtra seed blends compared to 100% ref uge 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). Thes e differences were statis tically significant at two out of three locations. The a pplicant attributed these differenc es 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 plan ts 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 s eed 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 plan ts, no statistically significant differences were found. Damage ratings pooled for both hybrids ra nged 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 economica lly 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 fi ve 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 we re 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. E ach treatment had 5 identified plan t clusters that were chosen for infestation purposes. Injury fr om CEW feeding was scored appr oximately three weeks after the infestation date and assessed by me asuring the total square centimeters of damage on ear tips and kernel area. Additionally, the num ber of live CEW found in a five-plant cluster was recorded at the time of scoring.
ANOVA was used to analyze the main effects an d 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 differenc e 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 plot s (statistically significant). Dama ge in seed blends ranged from 2.8 to 13.4 cm 2 /plant. Damage in refuge plants fr om isoline plots ranged from 5.1 to 17.0

cm 2 /plant. The difference in larvae/ear from the tw o 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 pl ant in seed blends (immediately adjacent and two plants removed) wa s compared to the efficacy of Bt plants in pure stand Bt plots. No significant differences be tween the different types of Bt plants were detected. The mean number of la rvae/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 betw een the Bt plants (adjacent and two plants removed from refuge plant) in seed blends, no st atistically significant differences were detected. Damage ranged from 0.1 to 2.0 cm 2 /plant, and mean larvae/ear ranged from 0 to 0.6.

Pioneer/DuPont Conclusions: “Data from this study show that maize cont aining a pyramid of 1507 and MON810 provided very good efficacy against moderate to high manua l infestations of CEW. These results also demonstrate that OAM Xtra maize plants in close pr oximity 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 differe nces 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, spatia lly explicit, and consis ted 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 cell s in each field were designated to have 5% nonBt maize. Simulations were run for 400 generatio ns (200 years). A torus was simulated so that adults moving off of one side of th e 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 33333 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 plac ement 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 w ith refuge moved yearly (see Figure 1 above)

Scenario C : 5% blended refuge

- 18 -Assumptions and Parameters :
Pioneer/DuPont evaluated the durab ility of OAM by investigating a set of worst-case as well as benchmark parameters for European corn borer and southweste rn corn borer (see Table 3 ). Benchmark parameters represented what the app licant 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 repr esented values of uncer tainty for which there was less empirical support or published data were scarce. The applicant stated that dominance values were estimated based on empi rical data testing of homozygous susceptible and resistant, as well as heterozygous re sistant insects from Cry1F and Cry1Ab resistant colonies. It wa s 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 (resistan t 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 publis hed literature. Other survival parameters were incorporated such as egg-to-neonate survival rate (0.9025), pre-disper sal tasting, and overall survival to the late instar stage, carrying capacity, as well as overwintering survival.

Benchmark values for cross-resistance were se t 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 (ra ndom) mating took place. For SWCB, only 1% of males were allowed to move before mating occurre d; 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 even t. 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 1 st 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 1 s t instar leaving refuge plant 0.76 0.76

Probability of 1 s t 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 we re verified by manual calculation. The verified matrices included the dominance value, cross re sistance, mating frequency, survival rate, and oviposition.

Results from the current Pioneer /DuPont model were verified ag ainst 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 pr ocess. 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/DuP ont’s model. The results be tween 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 conduc ted for the several parameters displayed in Table 4 below. A parameter was determined to be se nsitive 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 para meters 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- 4 years. Coefficient of dominance (fold difference between vegetative and reproductive maize) 1-5 fold

Pre-dispersal tasting survival 0.4-0.7
Survival of susceptible 1 s t instar leaving Cry1Ab or Cry1F 0.0001-0.05

Probability of 1 s t instar leaving Bt 0.7-0.99
Probability of 1 s t instar leaving refuge plant 0.4-0.9

1 s t

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 la te 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, Extirpation occurred in 3yrs. Coefficient of dominance (fold difference between 1 st 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 1 s t instar leaving Cry1Ab or Cry1F 0.0001-0.05

Probability of 1 s t instar leaving Bt 0.7-0.99
Probability of 1 s t instar leaving refuge plant 0.4-0.9

1 s t

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 locat ion) and seed blend modeling, the following parameters were determined to be sensitive (a n apparent reduction in durability): initial resistance allele frequency, dominance, and cr oss-resistance. In addition to these three parameters, late instar survival and movement, bl ended 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 suscepti ble population to build up year after year. For block refuge simulations with the refuge location moved year ly, cross-resistance, initial resistance allele frequency, and dom inance were sensitive parameters.
For seed blends, cross-resistance, initial resistance allele freq uency, 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 assumpti ons 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 o ccur primarily in the native field. A structured refuge that is maintained in the same location year after year would th en create a sink of susceptible insects over time. However, the appl icant 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 popula tion extinction was reached by 5.5 years when refuges were moved yearly. Seed blen ds were predicted to have a durability of 16 years under worst-case assumptions.

- 24 - Pioneer/DuPont stated that a key consideration wa s whether refuge plants in a Bt seed blend produced sufficient amounts of susceptible insect s to effectively delay resistance evolution. Theapplicants concluded that the model accurately pr edicted a reduction in “effective refuge” from a seed blend (lower production of su sceptible 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 populat ions 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 w ith 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 th e risk that was historically associated with blending lepidopteran refuge fo r single trait high-dose products . The approval of a blended approach to refuge deployment will signifi cantly reduce the complexity of planting and management of refuge acres and will have a positive impact on refuge compliance. Harmonizing refuge deployment for stacked lepi dopteran 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 benef its not only the grower (by reducing planting complexity and refuge ma nagement) but the public as well, by ensuring 100% compliance, thereby extending the lifespan of more environmenta lly 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 es tablishing successfully on refuge plants in pure plots as well as in seed blends was low. So metimes 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 ge neration 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 additiona l 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 numb ers on refuge plants in seed blends from second generation infestation trials likely repr esent an overestimate because second generation adults may be less likely to oviposit onto refuge plants in 5% and 10% s eed blends than they would otherwise do in a structur ed refuge configuration. The SA P (2010) stated that while ECB adults cannot distingui sh 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 s eed 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 Pione er/DuPont, could in some cases be even lower than 60% (max. estimated) compared to emergen ce 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 (1 st gen) on Bt plants surrounding refuge plants in seed blends was 0 insects/plant (see Table 2 ). This is not surpri sing considering that establishment of larvae on refuge plants was low. Establishment for 2 nd generation ECB on refuge plants in seed blends was greater, and hence, the n umber of larvae encountered on surrounding Bt plants increased correspondingly. B PPD concludes that these preliminary data are not sufficient to draw final conclusions abou t larval movement and survival from refuge plants onto surrounding Bt plants in OAM (and OAM Xtra) seed ble nds. 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 locatio ns, and natural infesta tion was sparse; only few larvae established on refuge plants in seed ble nds. Hence, it is not surp rising that little or no movement was observed onto surrounding Bt plants. Dispersal of later instar larvae cannot be adequately addressed due to th e limited number of SWCB that established on refuge plants. Pioneer/DuPont’s preliminary da ta should be used and inte rpreted with caution. BPPD recommends that additional trials (multi-year, multi-location) be conducted and data be collected to better estimate the propensity of la rval movement and survival for SWCB. Pioneer/DuPont reported that in some cases se ed blends caused less susceptible SWCB to emerge from refuge plants relative to structur ed refuges of equivalent non-Bt proportions. BPPD believes that a reduction in effective refuge s hould be expected with a seed blend strategy,
- 26 -especially for a pest that has a great larval m obility (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 incorpor ate 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) surroundi ng refuge plants; BPPD notes th at this is not surprising considering that natural infestation levels were low. Under high infestat ion levels, damage to surrounding Bt plants might have occurred. The a pplicants 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 ble nd plots may be functional but not equivalent refugia compared to structured refugia of similar non-Bt proportions due to a reduc tion in effective refuge (as discussed above). Hence a 5% OAM blend shoul d 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 eq uivalent to that of a 5% OAM structured block refuge. In the applicant’s artificial infestation experi ments 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 th is implies that a reduction in effective refuge from a seed blend implementa tion of OAM can also be expected for CEW. BPPD noted that the CEW establishment was lo wer on refuge plants than on Bt plants and ranged from 0.1-0.4 (mean) larvae/ear on non-Bt pl ants and from 0.1-0.7 (mean) larvae/ear on Bt expressing plants (7 out of 10 plants sampled had a larvae. Pi oneer reported that there was no statistically significant difference between Bt plants in blends a nd pure stands. Equally important though, the numbers of CEW recovere d from Bt corn ears was higher than on non-Bt plants; the observed ear feeding damage was, however, mu ch lower on Bt than on non-Bt corn. No discussion was provided by the app licant 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 efficac y 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 ref uge plants was significantly reduced compared to pure refuge plots. BPPD conc ludes 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 m odel structure, assumptions, parameters, and a discussion about outputs, sensitivit y analyses, and their own interp retations, which facilitated the Agency’s review of the applic ant’s submission. The following sect ion summarizes and discusses

- 27 -BPPD’s concerns and recommendations with respect to some assumptions and parameter values chosen by Pioneer/DuPont and contains recommenda tions 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 appl icant’s approach of m odeling larval dispersal explicitly. This allowed Pioneer /DuPont to incorporat e dynamics of seed blends that emerge from plant-to-plant movement that would otherw ise 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, th eir simulations were run out pa st 200 years when local pest population densities in the model were below one individual/hectare. This introduced the increased importance of genetic dr ift, which is a stochastic pro cess. Hence, BPPD recommends that for simulations where populations are expected to have very low densities that it might be beneficial to switch to a stochas tic rather than deterministic mode l. It appears counterintuitive to run simulations for so many hypothetical generations when extinction is pract ically 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 appli cant’s approach could be justified. Another approach to modeling the durabilit y of OAM could be to artificia lly inflate the fecundity (many fold) of the pest to a point where extinction ceas es to occur. Then the model outputs for a 5% blend and 5% structured refuge could be compar ed 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 initi al 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 fr equency for ECB was set at 0.01 (best case) and 0.05 (worst case). BPPD concl udes that these assumptions seem reasonable based on 2010 SAP discussions (SAP 2011). It wa s not surprising that this ke y 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 es timates. No such FRAF were reported in Pioneer/DuPont’s submission to th e Agency for this application.

• Dominance : Pioneer/DuPont stated that they obtained their estimates for ‘ h ’ by empirically testing individuals (SS, RS, a nd RR) from Cry1F and Cry1A resi stant 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 (w orst-case) and 0.02 (best) and 0.1 (worstcase), respectively, yet incor porated 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 coul d 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 re ported that dominance based on LC 50 was estimated at ‘ h ’ = 0.27 for reproductive stage plants suggesting th at resistance to Cry1Ab was intermediate and incompletely recessive compared to other reports in the literatu re. 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 resist ance functionally recessi ve 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 fi eld conditions (Crespo et al. reported that resistance in their lab selected co lony was polygenic and controlled by > five loci). However, in light of these data, BPPD recommends th at Pioneer/DuPont consider greater dominance values for ECB and SWCB worst-case simu lations on reproductiv e 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 sele ction of parameter values was based on Peirera et al. (2008) who demonstrated that resistance in their laborat ory selected ECB strain was recessive. When RR, RS, and SS-genotypes were exposed to Cry1F expressing corn plants, heterozygous resistant in dividuals exhibited survival rates similar to susceptible individuals; however, this similarity was stronger on reproduc tive maize than vegetative maize (which is the opposite of what was reported for Cry1Ab co rn). The authors concluded that resistance was functionally recessive but more recessive for reproductive than vegetative Cry1F corn. Based on percentage of larval survival, percenta ge of weight gain, and survival and weight gain combined, their reported dominance va lues for vegetative corn were 0.23, 0.27, and 0.07, respectively. For reproductive corn, t hose 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 co rn. 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 gr owth 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 th e actual survivorship s of heterozygous individuals were for Cry1Ac and Cry1F in the mo del. 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 actu al RS-survival data used in the model.
The applicants reported that susceptible 3 rd instar mortality of SW CB (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) th ey 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 th ere 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 not es that if pre-dispersal survival is this high, that the dominance value should not be chos en to represent a high-dose but rather a low dose toxin. Pioneer/DuPont need to revise their modeling anal ysis to incorporate this correction. The applicant’s sensitivity analysis showed that dominance for pre-dispersaltasting survival was sensitive, especially at upper values around 0.2 (still too low for a lowdose effect on the target pests); BPPD expects th at the durability of OAM seed blends will be significantly affected after correcting the value fo r 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 th e pre-dispersal tasti ng mortality. Inclusion of this parameter addresses the BNI m ovement hypothesis (SAP 2011). However, the applicant’s model lacks inclus ion of the second toxin mortal ity pathway associated with movement: the NBI hypothesis addresses mortal ity associated with RS genotypes that originate and remain during most of their de velopment on non-Bt but move as late-instars onto Bt where they have a certain probabil ity of survival. BPPD recommends that Pioneer/DuPont incorporat e this particular larv al movement scenario/mortality for ECB and SWCB into their model. BPPD recognizes that there are uncertainti es 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 approx imately 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 pl ant 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. Grea ter 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 w ithin 800 meters of the location that they mated. For ECB, it was assumed that 100% of the females disper sed and oviposited in such a manner. BPPD notes that there is some uncerta inty 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) obser ved that males engaged in a pr eferred pre-copulatory flight behavior within their natal fields. Hence, BPPD concurs with Pion eer/DuPont’s assumption that SWCB males mate primar ily 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 densitie s. “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 ovipos ition (egg dispersal) of ECB in seed blends and stated that a large body of ev idence suggested that Bt protein in maize did not affect ECB ovipositional behavior (He llmich 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 ble nded 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 structur ed refuge strategies. (SAP 2011)

BPPD recommends that Pioneer/DuPont includ e a degree of non-random oviposition for first generation as well as second generation ECB and SW CB 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 lo cus by a gene at another locus. The SAP (2011) recommended that different mechanisms of ep istasis be considered for products expressing pyramids of two or more toxins. As was disc ussed 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 im pacts 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 imp acts 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 (CR 1 Æ 2 ) for their best and worst-case scenar ios, which was determined to be sensitive in the applicant’s analyses. Cross-resistance is one form of ep istasis that the SAP recommended including for those particular Cry-toxins. BPPD recommends that a low level of cr oss resistance between Cry1F and Cry1A (CR 2 Æ 1 ) 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 bl otting 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 ont o Bt. The SAP mentioned that this may be a more likely mechanism for thes e 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. B PPD notes that if the ap plicant 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-w ise regression, standard regression, Bayesian model analysis, etc). In addition, a probability analysis would also allow the registra nt 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 comparis on 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 expre ssed in the pyramid (OAM) and re ported that no sensitivity was apparent. BPPD notes that it would be more rele vant 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 explori ng survival of RS-indi viduals that is in excess of 5x the survival for SS-individuals.
d) Pioneer/DuPont Results The applicant estimated that for ECB, the durab ility of OAM would last more than 200 years under best-case assumptions with a seed blend or structured refuge st rategy. BPPD concludes that it is insufficient to report “greater than”-re sults 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 as sumptions for ECB, the estimated durability for a structured refuge and seed blend was 35 year s 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 signifi cant or whether the applicant wa s 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 dete rmine 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 durab ility 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 th an”-results because actual numbers of predicted durability should not be taken at face value and a relative comp arison between actual “durability years” may be more meaningful. When Pioneer/DuPont used wo rst-case parameter assumptions, the estimated durability for blocks was still >200 years when th e 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 sw itching from a block to a seed blend strategy under worst case assumptions using the applicant’ s model construct. BPPD reiter ates 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 stru ctured refuge simulations after three years for ECB and four (and five) years for SWCB. The a pplicant discussed the importance of genetic drift at this point and also adde d 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 effectivel y delay resistance evolution, the number of insects emerging from refuge plants in OAM seed blends were suffici ent to provide a durabilit y 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 occu r 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 proce ss 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 exte nsive theoretical analysis us ing 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, dos e, epistasis, adult and larval movement). The SAP demonstrated that assuming moderate lepidopteran larval movement and density-dependence, effectively no seed blen d percentage provided durability equivalent to that of the structured refuge with equivalent non-Bt refuge. The result s still represented an overestimate of seed blend durabi lity because the Panel did not incorporate selection in the corn ears and explore larval movement and epistasis sufficiently. The Pane l stated that in addition to seed blends (with high-dose toxi ns causing low RS-survival) bei ng less durable than structured
- 33 -refuges with the same non-Bt percentage, seed bl ends 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 he terozygote survival. The SAP concluded that based on the uncertainties and their modeling anal ysis that at the moment, a seed blend for lepidoptera was not scientifica lly supported. BPPD notes that th e 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 ble nds 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 applican t’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% structur ed refuge. All models (whether complex or simple) are idealizations and abst ractions of reality, have inco rporated 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 es timates’ and in some cases ‘best guesses’ and do not represent actual values encountered in nature. The SAP (2011) therefore recommended that scie ntists and foremost regulatory agencies should pay more attention to relative comparisons between vari ous 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 mode l scenarios so that a percent difference can be determined between various st rategies and make the recommended changes to their model as discussed throughout th e 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 applican t 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 an alysis would allow the Agency to assess the durability of a greater blend percentage. Generall y, BPPD would expect a 10% seed blend to be more conservative and more durable than a re spective 5% seed blend with two lepidoptera toxins. Carriere et al. (2004) concluded that di fferences in Bt durability predicted by block and seed blends (caused by a reducti on 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% bloc k with pyramid) can be made. The SAP (2011) demonstrated that a dual gene, hi gh efficacy product with a seed blend approach targeting ECB and SWCB was less durable than a block refuge of equi valent non-Bt proportion. It was unclear however from thei r 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 migh t 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 wa s switched from a 5% st ructured 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 deficienci es with Pioneer/DuPont’s model submission for OAM, and BPPD is unable to conf idently assess the risk of resi stance 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 ma y 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 durabil ity for a seed blend and structured refuge.

• Incorporate a probability anal ysis to address uncertainti es 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 mo rtality/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 th eir development on non-Bt but move as lateinstars onto Bt where they have a certain probability of survival.

• Focus IRM strategy on species wi th greatest uncertainties and risk of evolving resistance – in this case southw estern 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).

- 35 -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 Pr otected Crops”, Springer, 2004.
Crespo ALB, TA Spencer, AP Alves, RL Hellm ich, 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 European corn borers (Lepidoptera: Crambidae) infesting transgenic corn expressing Cry1Ab 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 (Lepdi optera: Crambidae) females. Environmental Entomology, Vol. 37: 1280-1290
Dorhout DL, TW Sappington, and ME Rice. 2011. F light behaviour of European corn borer infected Nosema pyrausta. Journal of Applied Entomology, Vol.135: 25-37
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Ives AR and DA Andow. 2002. Evolution of resist ance to Bt crops: directional selection in structured environments. Ec ology Letters, Vol. 5: 792-801
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Porter P. 2011. Email communication w ith JC Martinez, US EPA/OPP/BPPD.
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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, 200 0 on Bt plant-pesticid es risk and benefit assessments: insect resistance management. Re port 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 Optim um AcreMax1 corn rootworm-protected corn. Memorandum from J.E. Bailey (DFO) to Dr. D. Edwards (Director of OPP) on May 6, 2009.

- 37 -- 38 - EPA Scientific Advisory Panel. 2011. Transmitta l Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held D ecember 8-9, 2010 to Address Scientific Issues Associated with Insect Resistance Management for SmartSta xTM Refuge-in-the-Bag, a Plant-Incorporated Protectant (PIP) Corn Seed Bl end. Memorandum from Dr. Sharlene Matten (DFO) to Dr. Steven Bradbury (Director of OPP) on March 3, 2011.

BPPD, 2008a. Review of studies submitted by Pion eer 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) Pr oduct 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 Pion eer’s modeling addenda (MRIDs 478710-01, 478879- 01, and 479108-01) in support of thei r 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 co rn 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 mana gement (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. Reynol ds to A. Sibold dated October 20, 2010.