CONCLUSIONS AND RECOMMENDATIONS

Pioneer/DuPont addressed the reco mmendations made in BPPD (201 1): the applicant artificially inflated t he fecundity for European and southwestern corn borers to avoid population extinction in their modeling analyses

, included some worst

- case values for several sensitive parameters in their new simulations

, conducted a probability analysis to be able to report a risk profile to the Agency, and addressed the most germane uncertainties described by the SAP (2011) for seed blends with pyramided PIPs targeting Lepidoptera. However, Pioneer/DuPont did not include non

- unifor m oviposition by Lepidoptera in their main modeling analyses and explore greater female (southwestern corn borer) dispersal out of seed blends.

I. RISK CHARACTERIZATION

After careful review of the applicant’s new modeling submission, BPPD conclude s that :

**•** The 20% single toxin block refuges had the lowest durability estimates of all options tested in
Pioneer/DuPont’s model

. Those durability results were, however, artificially reduced by higher fecundity assumptions. Pioneer/DuPont estimated blends to be betw een 6 and 60

- fold more du rable than 20% blocks for single toxin PIPs.

**•** T he proposed seed blends (5% and 10%) will likely be substantially less durable than a 5% block refuge
for the same product (100% compliance assumed). This was al so concluded by the SAP (2011). With realistic assumptions of non

- compliance the estimated durability for the block scenario would likely be
somewhat lower but still more durable than the blends. Pioneer/DuPont’s model estimated that a 5% block had close to double the durability of blends for both pests (benchmark assumptions). The durability of the pyramided blends is highly likely to fall in between the two block options. They can be

ex pected to be more durable than the single toxins (especially under assumptions of low cross

r e sistance ). The uncertainties described below ( effects of increased fecundity, no model ing of non

uniform oviposition and greater dispersal out of blends) can be expected to further erode the durability
of blends.

**•** A 10% blend is likely to be more durable t han a 5% seed blend for the same product. Pioneer/DuPont reported, however, that the lower blend percentage had slightly greater durability than the higher blend percentage. Possible explanations for this unexpected outcome are discussed in the uncertainti es section
below.

**•** These conclusions are based on all available information, including Pioneer/DuPont’s modeling, the 2011 SAP report, and EPA/ORD modeling analyses created for other seed blend products. Quantification of the risk (i.e. level of durability) for Optimum AcreMax and Optimum AcreMax Xtra is complicated by uncertainties in some biological parameters including uniform/non

- uniform oviposition, effects of inflated fecundity, degree of female pre

- ovipositional dispersal as well as aspects of the

pro bability analysis. All these uncertainties together could functionally lower the durability of seed blends relative to block refuges.

**•** In consideration of the cumulative effect of the uncertainties and w itho ut additional modeling , BPPD is
unable to complete ly quantify the relative durabilities of the various refuge options . BPPD expects, however, the relative durability relationship to hold true: 5% pyramid block > 10% pyramid seed blend > 5% pyramid seed blend > 20% single toxin block

.

**•** The 10% Optimum Acre Max 1 seed blend (expressing Cry34/35) targeting corn rootworm was approved in 2009. The applicant seeks to combine this single CRW gene with the pyramid PIP for Lepidoptera into a new 10% seed blend product (Optimum AcreMax Xtra). As stated above, the ris k of- 3

- resistance development and relative durability cannot be quantified for any of the proposed blends; however, BPPD expects the risk of resistance evolution to be lower for a 10% seed blend expressing two pyramided PIPs than a 5% blend as proposed for t he Optimum AcreMax product targeting Lepidoptera only.

II. UNCERTAINTIES :

BPPD has identified the following uncertainties in Pioneer/DuPont’s analysis:

1) Non

- uniform oviposition behavior was not included into Pioneer/DuPont’s benchmark simulations although a sensitivity analysis for this parameter showed a trend of greater durability for the 10% seed blend over the 5% blend when conservative, yet realistic, assumptions of non

- uniform oviposition favoring Bt plants were included. Based on the advice received by the SAP (2011) as well as information published in Hellmich et al. (1999), BPPD concludes that any durability analysis of a seed blend application targeting lepidoptera should conservatively incorporate non

- uniform oviposition in
order to address situatio ns where greater insect damage to refuge plants in a seed mixture could result in
greater oviposition onto Bt plants

. One of Pioneer/DuPont’s proposed products (Optimum AcreMax Xtra) contains a corn rootworm protectant gene; for PIP plants that receive add itional below ground protection, early crowding out of refuge plants by Bt plants is another reason why non

- uniform oviposition needs to be addressed in modeling analyses.

2) Pioneer/DuPont’s durability estimates for the 5% seed blend were greater than for the 10% seed blend

s trategy . BPPD determined that this non

- intuitive result may have been a consequence of having inflated the fecundity of the target pests by 6

- and 4

- fold for ECB and SWCB

. This unexpected outcome of greater durability for a blend contai ning a lower non

- Bt proportion was also the topic of discussion in the SAP report (2011) and Ives et al. (2011). It was demonstrated there that by inflating fecundity greatly that a two

- toxin

- pyramid in a seed blend with low non

- Bt proportions could be dri ven to have increased durability over a blend with greater non

- Bt proportions when target pests were mobile

. I n fact, the SAP demonstrated that at first the durability of seed blends increased with increasing proportion of

non

- Bt plants. Then a precipice w as reached were durability began to decrease for blends with greater proportions of non

- Bt plants. As fecundity was successively inflated, the precipice moved towards lower
values of blends with non

- Bt proportions ( Figure 1 ). In ot her words, when fecundity was greatly increased, the effect was that there was greater mortality of susceptible genotypes as a result of density

dependence; this led to greater selection of resistant genotypes (Ives et al. 2011). “ Because all sorts of
vari ability are not considered in deterministic simulations, it is possible to push the model over the precipice, which mark s the point on the graph where populations beg i n to head towards extinction and the density

- dependent model bec o me s density

- independent; accelerated resistance evolution can be observed at this point; the precautionary advice is to stay away from this precipice or boundary ” (Ives, personal communication).

Since the applicant’s durability estimates appear to be derived from beyond this bo undary (precipice),
BPPD conclude d that it might be more meaningful to look at the sensitivity analysis conducted for non

uniform ovi position: when Bt plants were favored as oviposition sites , the fecundity of susceptible
genotypes may have been indirectly reduced (of ECB but not so for SWCB) and density

- dependence
moved Pioneer/DuPont’s model back into the realm where durability estimates conformed to expectations under resistance evolution models and where seed blends with greater non

- Bt proportions had g reater durability than those with fewer non

- Bt plants

. Modeling analyses conducted in the past by EPA/ORD also predicted that a greater percentage (i.e. 10%) of refuge plants in a seed blend will be- 4

- more durable than a lower percentage (i.e. 5%) of refuge plants (BPPD 2010a and BPPD 2010b). BPPD
also considered Carriere et al. (2004) who reported that differences in Bt durability predicted by block
and seed blends of equal refuge plant proportion s (caused by a reduction in refuge insects in seed blends) cou ld be overcome by increasing the proportion of non

- Bt plants in seed mixtures.

3) Pioneer/DuPont conducted a sensitivity analysis for the “number of eggs produced in natal fields” by SWCB in block refuges (single and pyramided PIPs) but not for seed blend sc enarios. The applicant reported that durability results for blocks were not very sensitive to changes in this parameter although a slightly decreasing trend in durability was noted for single PIPs as pre

- ovipositional dispersal increased

. For a 5% block re fuge with a pyramided PIP (where no resistance evolved for the duration of the
simulation

– 400 yrs), the applicant reported that the final resistance allele frequency was slightly higher when there was more dispersal out of the fields. This result agrees with what the SAP (2011) reported, namely, that with high female dispersal, resistance would evolve faster. Pioneer/DuPont did not compare the durability of blends to blocks when varying this parameter. This w ould have been an important
relative comparison , since dispersal in blends requires adults to only move short distances away from the refuge plant before having dispersed from their “natal field”. Hence the effects of female pre

ovipositional dispersal out of seed blends can be expected to have a great er effect on resistance evolution in seed blends than block refuges. The SAP (2011) stated that “for seed mixtures dispersal rates will be 100%, and greater than between field refuges. The main consequence [of greater dispersal rates] is that seed mixtures will have faster resistance evolution than between field refuges”.

III. RECOMMENDATIONS :

It is recommended that Pioneer/DuPont incorporate the following into their model: a) Non

- uniform oviposition for both generations of lepidoptera in a 10% seed blend (OAM Xtra); b) Non

- uniform oviposition for the second generation of lepidoptera in a 5% seed blend without a corn
rootworm trait ; and c) Increased female (SWCB) pre

- ovipositional dispersal out of seed blends.

It is recommended that Pioneer/DuPont further clarify the following about the modeling : d) How was fecundity increased in seed blends? Does the 6

- fold ( and 4

- fold ) increase refer to the fecundity of susceptible insects on refuge plants r e l a t i v e to fecundity on Bt plants? e) How was uniform oviposition dealt with in the model code? The methodology should be explained. Do adults form one big random mating po ol in seed blends

, and eggs are uniformly applied across the field (or else)? f) How was non

- uniform oviposition dealt with in the model code o f t h e s e n s i t i v i t y a n a l y s i s ? Pioneer/DuPont should explain the methodology used.

BPPD recommends research on: g) O viposition al behavior in seed blends under , for example, increased pest pressure ; h) The potential and degree of crowding out refuge plants by Bt protected plants in blends expressing corn

rootworm tra its under different environmental conditions; i) 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).- 5

- I. BACKGROUND

BPPD reviewed biological data and simulation modeling for lepidoptera pest of corn that was submitted by
Pioneer/ DuPont to support a registration request for a 5% O ptimum AcreMax (expressing Cry1F and Cry1Ab)
and 10% Optimum AcreMax Xtra RIB (expressing Cry1F, Cry1Ab, a nd Cry34/35) (BPPD 2011a)

. In that review, BPPD identified a number of concerns and deficiencies wi th the proposed refuge strategies and was unable to quantify the relative durability of the proposed seed blends (BPPD 2011 a ). The following is a summary of the deficiencies and concerns listed in the Agency’s review and deficiency letter sent to the appli cant (BPPD 2011b) :

BPPD required that Pioneer/DuPont incorporate the following changes into their model, provid e a comparative modeling analysi s, and address some of the remaining biological uncertainties.

**•** Report final resistance allele frequency and actu al estimates of durability rather than ‘greater than’ because relative comparisons between various strategies may be meaningful even if actual numbers are

not.

**•** Artificially inflate fecundity of the pest to 1) avoid extinction, 2) avoid introducing the impo rtance of genetic drift, and 3) determine relative durability for a seed blend and structured refuge.

**•** Incorporate a probability analysis to address uncertainties with various important parameter values and to provide the Agency with a risk profile (i.e., 0 %, 5%, and 50%) for Optimum AcreMax and the modeled IRM strategies as well as a level of significance between estimated durability for different strategies.

**•** Provide comparative modeling (incorporating B PPD’s recommendations ) to evaluate:

- 20% block refuge ( single toxin)

- 5% block refuge (pyramid)

- 5% blended refuge (pyramid)

- 10% blended refuge (pyramid)

**•** Correct the dominance value for pre

- dispersal mortality/survival to be reflective of a low

- dose exposure rather than a high

- dose exposure.

**•** Include non

- uniform oviposition parameter for first generation lepidoptera in Optimum Acre M ax Xtra and second generation adults in Optimum AcreMax seed blends.

**•** Include NBI

- movement hypothesis (SAP 2011) to address mortality associated with RS genotypes that originate and rem ain 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 fe eding 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).

On June 27, 2011, Pioneer/Du Pont responded to the Agency’s review by resubmitt ing additional explanations and modeling ( M R I D 4 8 5 2 1 9

- 0 1 ) to address the uncertainties and deficiencies noted in BPPD (2011 a , b ). The following review reflects BPPD’s summary of the applicant’s re submission and the Agency’s ana lysis thereof

.- 6

- II. PIONEER REVISED MODELIN G

Applicant’s Methods:

Pioneer/DuPont’s model was deterministic, spatially explicit, and consisted of a grid 100 m x 100 m cells (each cell = one hectare); simulations occurred on 400 cells for a total of size o f 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. The longest distance between refuge areas of each field was limited to < 600m; this placement favored block refuges and assumed random mating for ECB. Block refuges were all placed on the same short side of the ten rectangular fields and kept fixed at that location year after year (modeling scenario A) or switched to the opposite side each consecutive year (modeling scenario B). To simulate a 5% blended refuge, all 40 cells in each field were designated to have 5% non

- Bt maize. A torus was simulated so that adults moving off of one side of the system arrived on the other side.

Pioneer/DuPont adjusted the previously chosen values for cross

- resistance, fecundity rate, dominance, and proportion of eggs produced in the natal field. New benchmark and worst

- case parameter values are provided in
Tables 1 and 2 below.

T he dominance value for Cry1Ab and Cry1F were increased. For Cry1Ab vegetative stage maize the new

worst

- case value was set at to 0.27 for reproductive stage maize. B eca use of the assumption that there is a two

fold difference between dominance values of reproductive and vegetative stage maize, a new dominance value for vegetative stage maize was also calculated (0.27/2=0.135). For Cry1F the dominance value for vegetative stage maize was increased to 0.23 and 0.1 for reproductive stage maize. The new coefficient of dominance was calculated based on these new values (0.1/0.23=0.435).

To avoid extinction in the majority of fields and the impact of genetic drift (as a resul t of low population densities), the applicant increased fecundity of ECB and SWCB by 6

- fold and 4

- fold, respectively.

Additionally, pre

- ovipositional dispersal was increased for SWCB female s by decreasing the benchmark
parameter value “Proportion of Eggs Produced in Natal Field” from 0.75 to 0.5 ; the sensitivity of this parameter

was explored for a range of values (0, 0.1, 0.25, 0.4, 0.5, and 0.75)

.

The duration of the deterministic model runs was extended from 200 years to 400 years. For IRM scenarios

w here resistance did not evolve within the set time limit, final resistance allele frequencies were reported. Resistance was assumed to have evolved when the resistance allele frequencies for both loci had reached 0.50

. Pioneer conducted a sensitivity analy ses for ‘ non

- uniform oviposition ’ in seed blends. The impact on durability
of blends from different oviposition ratios was explored (favoring Bt over non

- Bt 1:1, 2:1, and 4:1) (see Table s 3 and 4 )

.

Pioneer/DuPont conducted a probab ility analysis using a stochastic module written in R statistics software and provided estimates for 0%, 5%, and 50% level of risk that resistance would evolve within a certain time. The

PERT

- distribution was skewed toward worst

- case parameter values to ad ditional conservatism. Refuge blocks were randomly placed between two locations

. A total of 100 0 runs were conducted for each simulation. Parameters used in the probability analysis are displayed in Table 5.- 7

- The following refuge scenarios were modeled u sing the probability analysis:

**•** 1507 and MON810 single toxins with a 20% block refuge ( moved randomly ) every year;

**•** 1507xMON810 with a 5% block refuge ( moved randomly ) every year;

**•** 1507xMON810 with a 5% blended refuge; and

**•** 1507xMON810 with a 10% blended refu ge.

Table 1 . Benchmark and Worst

- Case Parameter Values for Revised Modeling of ECB Resistance to Optimum AcreMax

Changed

Parameters Original Model:
Benchmark Revised Model:

Benchmark Original Model:
Worst

- Case Revised Model:

Wo rst

- Case

Cross

- resistance of Cry1Ab  Cry1F 0.2 0. 2 0.3 0.3

Cross

- resistance of Cry1F  Cry1Ab 0 0.1 0.1 0.1

Fecundity (eggs/female) 288 1728 288 1728

Dominance of R

- allele
for Cry1Ab (vs) 0.01 0.01 0.05 0.135

Coefficient of Dominance for Cry1Ab 2

- fol d 2

- fold 2

- fold 2

- fold

Dominance of R

- allele
f o r Cry1F (rs) 0.01 0.07 0.05 0.23

Coefficient of Dominance for Cry1F 2

- fold 0.435 2

- fold 0.435

Dominance value for

pre

- dispersal tasting survival (spdt) 0.01 0.5 0.05 0.5

Proportion of eggs produced in nata l field 0 0 0 0

Table was modified from Table 1 in P ioneer/DuPont ’ s submission, p.10 ( M R I D 4 8 5 2 1 9

- 0 1 )

1 These were the original benchmark parameter values as referenced by BPPD in their risk assessment dated May 18 th , 2011.- 8

- Table 2

. Benchmark and W orst

- Case Parameter Values for Revised Modeling of SWCB Resistance to Optimum AcreMax

Changed

Parameters Original Model 1 : Benchmark Revised Model:

Benchmark Original Model:
Worst

- Case Revised Model:
Worst

- Case

Cross

- resistance of Cry1Ab  Cry1F 0.1 0.1 0. 3 0.3

Cross

- resistance of Cry1F  Cry1Ab 0 0.1 0.1 0.1

Fecundity (eggs/female) 441 1764 441 1764

Dominance of R

- allele to Cry1Ab (vs) 0.02 0.02 0.1 0.135

Coefficient of
Dominance for Cry1Ab 2

- fold 2

- fold 2

- fold 2

- fold

Dominance of R

- allele to Cry1F (r s) 0.02 0.07 0.1 0.23

Coefficient of Dominance for Cry1F 2

- fold 0.435 2

- fold 0.435

Dominance value for

pre

- dispersal tasting survival (spdt) 0.02 0.5 0.1 0.5

Proportion of eggs produced in natal field 0.75 0.5 0.75 0.5

Table was modified from Table 1 i n Pioneer/DuPont ’ s submission, p.10 ( M R I D 4 8 5 2 1 9

- 0 1 )

1 These were the original benchmark parameter values as referenced by BPPD in their risk assessment dated May 18 th , 2011.

Table 3 . Effects of ECB non

- uniform oviposition on the durability o f diff erent seed blend percenta ges

Seed blend Percentage Gen1: 1:1
Gen2: 1:1 Gen1: 1:1
Gen2: 2:1 Gen1: 2:1
Gen2: 2:1 Gen1: 2:1
Gen2: 4:1

Years to Resistance Years to Resistance Years to Resistance Years to Resistance

5% 217 174 145 124

10% 199 183 178 162 Table was modified from Table 4 in P/DP submission, p.14 ( M R I D 4 8 5 2 1 9

- 0 1 ) *Highlighted sections are durabilities predicted under P i o n e e r / D u P o n t ’ s model construct with n o n

- u n i f o r m oviposition assumptions believed to be more conservative by BPP D Ratio 1:1; females oviposit eggs equally onto Bt and refuge plants Ratio 2:1; females oviposit 2/3 of eggs onto Bt and 1/3 of eggs onto refuge plants Ratio 4:1; females oviposit 4/5 of eggs onto Bt and 1/5 onto refuge plants

Table 4 . Effects of SWCB no n
- uniform oviposition on the durability of different seed blend percentages

Seed blend Percentage Gen1: 1:1
Gen2: 1:1 Gen1: 1:1
Gen2: 2:1 Gen1: 2:1
Gen2: 2:1 Gen1: 2:1
Gen2: 4:1

Years to Resistance Years to Resista nce Years to Resistance Years to Resistance

5% 2 32 263 300 310

10% 136 159 163 180 Table was modified from Table 4 in P/DP submission, p.14 ( M R I D 4 8 5 2 1 9

- 0 1 ) *Highlighted sections are durabilities predicted under P i o n e e r / D u P o n t ’ s model construct with n o n

- u n i f o r m oviposition assu mptions believed to be more conservative by BPPD Ratio 1:1; females oviposit eggs equally onto Bt and refuge plants Ratio 2:1; females oviposit 2/3 of eggs onto Bt and 1/3 of eggs onto refuge plants Ratio 4:1; females oviposit 4/5 of eggs onto Bt and 1/5 o nto refuge plants- 9

- Table 5 . Parameters used in Pioneer/DuPont's PERT distribution

Parameter ECB SWCB

Min Mode 1 Max Min Mode 1 Max

Cross

- resistance of Cry1Ab  Cry1F 0.1 0.2 0.3 0.05 0.1 0.3

Cross

- resistance of Cry1F  Cry1Ab 0.05 0.1 0.15 0.05 0.1 0.15

Initial R

- allele frequency for Cry1Ab 0.0005 0.001 0. 05 0.0005 0.001 0.005

Initial R

- allele frequency for Cry1F 0.005 0.01 0.05 0.0005 0.001 0.005

Dominance of R

- allele to Cry1Ab 0.005 0.01 0.05 ** 0.01 0.02 0.1

Dominance of R

- allele to Cry1F 0.005 0. 07 0.23 0.005 0.07 0.23

Survival on Cry1Ab of
SS larva

– early instar 0 0.001 0.01 0 0.001 0.01

Survival on Cry1F of
SS larva

– early instar 0 0.001 0.01 0 0.001 0.01

Survival on Cry1Ab of
SS larva

– late instar 0.01 0.025 0.2 0.02 0.05 0.2

Survival on Cry1F of
SS larva

– late instar 0.01 0.025 0.2 0.125 0.25 0.75

Table was modified from Table 1 in P/DP submission, p.12 ( M R I D 4 8 5 2 1 9

- 0 1 )

1 Mode of each parameter is the same as the benchmark val ue in Tables 1 and 2 above

. ** According to P i o n e e r / D u P o n t ’ s su bmission these max values should have adjusted to 0.135

Applicant’s Sensitivity Analyses:

Sensitivity analyses were conducted for the parameters ‘non

- uniform oviposition’ and ‘proportion of eggs produced in natal fields’. As oviposition for first and sec ond generation s of ECB became less uniform, the durability for both the 5% RIB and the 10% RIB decreased. However, the 10% RIB became more durable than
the 5% blend option when non

- uniform oviposition was included . This shift in greater durability from the 5% to the 10% blend was attributed to the fact that density

- dependence became less important as the overall population density declined. Pioneer/DuPont contended that although they explored the effects of non

- uniform oviposition, there was no evidence in the literature to support that ECB preferentially oviposit onto Bt (Hellmich et al. 1999; Pilcher and Rice 2001). This appears to be the reason why this recommendation was not incorporated into the actual benchmark simulations (BPPD comment).

When the num ber of SWCB eggs laid in natal fields was decreased for block refuge scenarios, the durability increased slightly, and the applicant determined that this parameter was not sensitive.

Applicant’s Results :

Pioneer/DuPont cautioned that inflating the fecund ity of ECB and SWCB to avoid extinction and effects of genetic drift, as recommended by BPPD, lead to unrealistically small durability estimates as was evident for the single toxin simulations. For single PIPs deployed with a 20% structured refuge, estimat ed durabilities were less than the actual time the products had been commercialized. T he applicant recommended that results be
considered relative to each other rather than viewed as absolute predictions

.- 10

- The applicant concluded that a 5% Optimum AcreMax RIB was more durable against ECB and SWCB than either single gene PIP deployed with a 20% structured refuge irrespective of the fact whether the refuge was kept in the same location each year or randomly assigned betw een two possible locations. When benchm ark and

worst

- case assumptions were considered , the 5% RIB scenarios were estimated to be between 7

- 36 fold and 4

- 6 fold more durable than the single toxins MON810 (Cry1Ab) and 1507 (Cry1F) , respectively, deployed with a 20% block refuge. When the same com parisons were made for SWCB under the same benchmark and worst

case assumptions, then the durabilities for a 5% RIB were 18

- 58 and five

- fold more durable than the single
toxins MON810 and 1507 , respectively

.

Furthermore, Pioneer/DuPont reported that a 5% MON810 x 1507 RIB was more durable than a 10% RIB using the same pyramided toxins. The applicant reasoned that this was not unexpected based on the inflated fecundity levels chosen and the resulting increase in density dependent mortality on refuge plants in scenarios with a higher blend percentage compared to a lower blend percentage and cited to discussions in Ives et al. (2011) and SAP (2011) for support. A 5% RIB targeting ECB was only moderately more durable than the 10% RIB, while
for SWCB the durabil ity decreased nearly by half when the blend percentage increased from 5% to 10%. The applicant had set the (per

- plant) carrying capacity at 18 larvae (Gen1) and 22 larvae (Gen2) for ECB and 1 larva (both generations) for SWCB and noted that if was difficul t to determine whether the differences in durability seen between the two species was only a function of the different carrying capacities chosen (and DD

- mortality) or whether differences in mating behavior could have also influenced the results.

The appl icant stated that increasing the dominance value for the “pre

- dispersal tasting mortality” value from 0.02 to 0.50 created a survival differential between SS and RS individuals that was not supported by the latest
research (manuscript in process)

.

The 5 % block refuge scenario had the greatest durability of all simulated refuge options . But the applicant noted that compliance was modeled at 100% and that any compliance less than that would decrease the durability of the block refuge. The difference in dur ability between the 5% block and 5% RIB scenario would therefore be
reduced if non

- compliance was considered.

Table 6 . Relative durability comparison between different IRM and PIP strategies ( incorporating BPPD's recommendations )

PIPs IRM Strategy Durab ility in Years

ECB SWCB

Benchmark Worst

- Case Benchmark Worst

- Case

1507 20% block

- fixed 6 2 5 2

20% block

- moved 6 2 4 2

MON810 20% block

- fixed 32 3 13 2

20% block

- moved 31 3 7 2

1507xMON810 5% block

- fixed 362 17 >400 * FRAF(Cry1A) =0.001009

FRAF (Cry1F) =0.001012 5

5% block

- moved 357 16 >400 * FRAF(Cry1A) =0.001101 FRAF(Cry1F) =0.001122 5

5% RIB 217 13 232 11

10% RIB 199 12 136 11 All block refuge comparisons assume 100% compliance ( M R I D 4 8 5 2 1 9

- 0 1 ) All scenarios were run with the assumption of uniform ovipos ition
Table was modified from Table 3 in P i o n e e r / D u P o n t ’ s submission, p.14 *When resistance did not evolve within the timeframe modeled, Pioneer/DuPont reported the final resistance allele frequencies

.- 11

- Table 7 . Probability analysis for relative duration of durability under different refuge and PIP strategies

PIPs IRM Strategy Durability in Years Different Probability Estimates for Resistance Evolution

ECB SWCB

0% 5% 50% 0% 5% 50%

1507 20% block

- moved 3 3 5 2 3 4

MON810 20% block

- moved 8 11 18 2 3 4

1507xMON 810 5% block

- moved 50 91 211 80 332 400

5% RIB 44 63 124 36 60 117

10% RIB 33 61 117 25 40 85
( M R I D 4 8 5 2 1 9

- 0 1 )

III. BPPD REVIEW

Pioneer/DuPont provided additional modeling simulations to address BPPD’s concerns expressed in the original review of their new product appl ication (BPPD 2011 a ). In this revised submission, the applicant focused on providing the Agency with simulations for an additional seed blend percentage (10%)

, which is directly relevant to the application for Optimum AcreMax Xtra maize (Reg. No. 29964

- R R ) that expresses both t w o Lepidoptera
and o n e Coleoptera Bt toxin . Additionally, the applicant submitted to the Agency simulation modeling for single gene PIPs ( the two toxins expressed in the pyramid) de ployed with a 20% block refuge

. All simulations
were run wit h several new parameter values as suggested by BPPD. During the original review of the applicant’s new product application (BPPD 2011a), an analysis of data and modeling showed that a 5% seed blend with a pyramid of Cry1Ab and Cry1F would be substantially less durable than a 5% structured refuge for the same product and likely more durable than a single gene product deployed with a 20% block refuge

. Since some scenarios were not provided at that time , however, BPPD was unable to quantify the relative gain o r loss in durability of a pyramided product deployed as a 5% seed blend compared to a 20% block and 10% seed
blend strategy.

BPPD’s discussion in this review will focus primarily on the relative compari son between the 5% and 10% seed
blend as well as between a 5% blend and 20% block refuge for a single PIP a n d the probability anal ysis conducted by the applicant

. In conclusion, BPPD will discuss the remaining uncertainties and revisit the SAP’s (2011) recommendations ( as discuss ed in BPPD, 2011) to determine w hether the applicant addressed those

concerns.

BPPD d iscussion of reported results :

For the following reported durability results derived with benchmark a n d w o r s t

- c a s e parameter value s , the applicant assumed that oviposition was uniform for both generations of lepidopt era. Other parameters that went
into the model to derive these results were listed in Table 1 and Table 2

.

SWCB results:
In Table 6 , Pioneer/DuPont report ed that the durabilities for a 5% and 10% blend targeting SWCB were much
lower than for a 5% block r efuge under benchmark assumptions . No resistance was reported after 400 years for a 5% block, and the final resistance allele frequencies had only reached 0.001 , while SWCB evolved resistance- 12

- in the blends in significantly less time. However, the 5% blend was more durable ( 1.7 times ) in these
si mulations than the 10% blend

. This is contradictory to the results reported by BPPD ( 20 10a ) that blends with
greater refuge proportions are more durable (although the simulations in that review were for a different t arget pest)

. This unexpected result of greater durability for the lower blend percentage is discussed in detail in the
uncertainties section below.

When Pioneer/DuPont used worst

- case parameter assumptions, the 5% and 10% seed blends had double the
durab ility of the 5% block and almost four times the durability compared to the 20% block deployed with a single gene. BPPD is unable to determine from the submission what parameter combination , aspect of model

structure, or other assumptions influenced the out come in which the 5% and 10% blend had equal durability
under worst

- case assumptions.

ECB results: When benchmark assumptions were used, Pioneer/DuPont estimated that the 5% block for the pyramid was 1.7
times and 1.8 times more durable than the 5% and 1 0% blend respectively. These lower durability results for greater ref uge proportions in blends are a consequence of the inflated fecundity numbers (as discussed in
uncertainties section ). The 20% block refuge was significantly less durable than the other th ree strategies. W hen oviposition was assumed to be non

- uniform in the first and/or second generation, then the durability for the 10%
blend was slightly higher than for the 5% blend and approximately 2 times lower than the estimated durability
of the 5% bl ock. The difference between the 10% and 5% blend would likely be greater with normal fecundity assumptions. The 5% blend was approximately 2.5 times less durable than the 5% block.

Probability analysis: The reported 0%, 5%, and 50% levels of risk report ed for ECB and SWCB were greatest for the single gene 20% block refuge, followed by the 10% seed blend, 5% seed blend, and 5% block refuge, respectively

, u n d e r P i o n e e r / D u P o n t ’ s m o d e l c o n s t r u c t

. BPPD recognizes and commends the effort that was put into this analysis by
Pioneer/DuPont. U nfortunate ly

, the actual risk profile is complicated by the overwhelming effect of increased fecundity on the results, which BPPD had recommended the registrant explore in order to avoid rapid (<10 yrs) population extinction in their prior modeling submission (see B PPD 2011)

. However, the larger point is that relatively speaking , single toxin PIPs, even with a 20% refuge, are less durable than pyramided PIPs ( under assumptions of low cross

- resistance ) deployed in seed blends with lower refuge proportions

.

Discu ssion of u ncertainties:

a) Uniform oviposition:

When Pioneer / DuPont presented their b e n c h m a r k a n d w o r s t

- c a s e durability estimates for a 1507 x MON810 RIB (see Table 6 ), they incorporated u n i f o r m oviposition o n t o Bt and non

- Bt plants for both generations of ECB and SWCB. Presumably this assumption was made based on a n article published by Hellmich et al. (1999). BPPD notes that this study analyzed results of different cage experiments in the field to evaluate 1)
whether adul t ECB could detect the presence or absence of differ ent Bt toxins (Cry1Ab, Cry1Ac) expressed in vegetative corn and 2) whether second generation adults would be deterred from laying egg masses onto reproductive refuge corn that had evidence of first genera tion infestation /damage

. Hellmich et al. found that
ECB adults did not select non

- Bt over Bt expressing maize in the first generation meaning that adults could not detect the expressed toxins in the genetically modified plants. The authors also stated that injuries to- 13

- reproductive (non

- Bt) maize from first generation larvae were not severe enough to affect oviposition of second generation adults. BPPD has concerns that in cases of greater pest pressure significant damage to refuge plants
in seed mixtures co uld occur and lead to non

- uniform oviposition behavior by ECB adults onto Bt plants. This

concern was also voiced by the SAP (2011) and discussed in BPPD’s review of Pioneer/DuPont’s original submission (BPPD 2011 a ). The SAP stated that this was more likel y a concern in seed mixtures (rather than block refuges) where Bt and non

- Bt plants were in close proximity. Additionally, 1507 x MON810 x Cry34/35 is designed to also protect Bt corn plants from below

- ground pests (such as corn rootworm), which could allo w Bt plants to outgrow (crowd out) unprotected refuge plants that incurred rootworm damage in such seed blends. In light of the uncertainties regarding uniform oviposition behavior for adult ECB (and SWCB), BPPD continues to support the recommendation of the SAP to incorporated non

- uniform oviposition for first and second generation lepidoptera when Bt maize receives protection from feeding damage of above and below ground pests and for second generation lepidoptera when protection in the blend is recei ved from damage of above ground pests only

.

Pioneer/DuPont did not include this recommendation into their benchmark and worst

- case simulations when reporting final durabilities for different IRM strategies. Their sensitivity analysis showed , however, tha t a 10% seed blend for ECB was relatively more durable than a 5% blend under moderately conservative assumptions of

non

- uniform oviposition . Whether the differences were actually significant was unclear. The sensitivity analysis for this parameter in SWCB did not show this same trend of increased durability with increased percentage (i.e.

10 %) of refuge plants in a blend

.

b) Increased fecundity and lower durability for the greater blend percentage:

When Pioneer/DuPont increased the fecundity for ECB and SWC B to avoid population extinction and the importance of genetic drift, as suggested by BPPD, they observed that under some circumstances (i.e. when oviposition was held uniform for ECB) the durability of the 10% blend was somewhat lower than t he durability
of the 5% blend d espite their being more refuge plants available and the potential for greater total refuge population in a 10% blend

. This behavior was also observed and discussed by the SAP (2011) as well as Ives et al. (2011)

. During the SAP meeting, Dr . Ives demonstrated in his presentation of a density

- dependent analysis
for a two toxin pyramid ( in a seed blend ) that for some special cases this result of having lower durability for a greater blend percentage may not be unexpected. In fact, Figure 1 (ex tracted from the SAP report) shows that at first the durability of the blend increases with increasing proportion of non

- Bt plants. Then a point (or precipice) is reached (see arrows) were durability begins to decrease for blends with greater proportions o f non

- Bt plants. As fecundity is successively inflated (see lower to upper graph), the precipice is moving towards lower values (see arrows)

. In other words, as fecundity increase s ( while all other parameter s are held constant), the speed of
resistance evo lution increases for the same proportion of refuge that lies past th is precipice

. When fecundity is increased, the effect is that there is greater mortality of susceptible genotypes as a result of density

- dependence; this leads to greater selection of resi stant genotypes (Ives et al. 2011). ” The precipice then marks the point on the graph where a population begins to head towards extinction and the density

- dependent model becomes d ensity

independent; the model behavior changes from what one would expect

, and accelerated resistance evolution can be observed at this point; the precautionary advice is to stay away from this precipice or boundary ” (Ives, personal communication). B PPD notes that the increasing fecundity for ECB and SWCB (6 and 4

- fold) may have mov ed Pioneer/DuPont’s simulated populations pas t this ‘precipice’ described by the SAP, which explains their decreased durability for a 10% blend compared to a 5% blend. This lower durability for higher blends is

t h e n l i k e l y a phenomenon due to inflating fecundity artif icially.- 14

- T h e a p p l i c a n t ’ s s e n s i t i v i t y a n a l y s i s s u g g e s t s t h a t w h e n assumption of uniform oviposition w e r e r e l a x e d a n d adults favored Bt plants over refuge plants 2:1 in the first and/or second generation, t h a t fecundity of the susceptible genotypes w a s r e d u c e d i n d i r e c t l y

. This had the effect of increas ing the durability estimates for the 10% blend over the lower blend percentage. This relative difference in durability was enhanced when oviposition o n t o B t w a s i n c r e a s e d for both generations of ECB

, which might be expected in a see d blend that expresses both corn rootworm and lepidoptera active toxins (see discussion above on ‘Bt plants crowding out refuge plants’). IRM analyses conducted in the past by EPA/ORD also predicted that a greater percentage (i.e. 10%) of refuge plants in a seed blend will be more durable than a lower percentage (i.e. 5%) of refuge plants (BPPD 2010a and BPPD 2010b).

Figure 1 . Generations to resistance failure (frequency of both alleles >0.5) for density

- dependent model for F= 100, 50, and 20 (labeled) when larval movement is 0, 0.3, and 0.6. Arrows mark the proportion of seed mixtures below which the population decreases towards extinction. Baseline parameter values are: S 1SS = S 2SS =0.01; h 1 =h 2 = 0.05; survival is divided equall y between pre

- and post larval movement; and initial allele frequency = 0.005. (This figure was extracted from the SAP report (2011) and modifie s Fig.A2

- 11 (p.134) to show the density

- dependent model runs only. Frequency dependent results were removed for t he purpose of this report.)

c) Probability analysis :

The applicant improved their original modeling analysis by conducting a probability analysis (PERT distribution) for both lepidoptera pests to address uncertainties with important parameters and to provid e the Agency with a risk profile for resistance evolution. BPPD noted that in Table 2 of the applicant’s submission- 15

- ( M R I D 4 8 5 2 1 9

- 0 1 ), the maximum values for dominance of the resistance allele for Cry1Ab was 0.05 (original benchmark) instead of 0.135 ( the newly adjusted value for current modeling submission ). The dominance value for the resistance allele is typically one of several sensitive parameters. It is unclear how much the risk profile
would change (if at all) had this maximum value been adjusted as outlined in the applicant’s report . The applicant also stated that they skewed the distribution of the PERT analysis towards the higher values for the purpose of deriving a conservative risk profile (but weighting the mean by a factor of four and there fore placing more confidence into that value). It is not clear to BPPD how skewing the distribution in this manner increased the probability of sampling the e x t r e m e value s ; it appears that the mean value w a s change d by skewing the
distribution . Also, Pionee r/DuPont did not state how many simulations were run to derive the ir probability
distribution. The SAP concluded that unless simulations were increased to close to 100,000 runs, it was unlikely
that extreme values were sampled from a predetermined range us ed to derive a PERT Beta distribution (SAP 2011)

.

d) Female dispersal out of natal fields :

Pioneer/DuPont conducted a sensitivity analysis for the “number of eggs produced in natal fields” by SWCB (one of the parameters for which BPPD suggested more conservat ive value s ) and reported that model results were not very sensitive to changes in this parameter . However, a slightly increasing trend in durability was
noted for single PIPs as the percentage of eggs in the natal (block refuge) field increased from 0% to 50% (analogous to decreasing durability with increasing pre

- ovipositional dispersal). For a 5% block (no resistance
evolved in 400 yrs), the applicant reported that the resistance allele frequency was slightly higher with more dispersal out of the fields c omp ared to when 50% of the eggs were laid in the natal field. This result matches up with what the SAP (2011) reported, namely, that with high female dispersal, resistance would evolve fast er

. Pioneer/DuPont did not compare the durability for blends to blo cks when varying this parameter. This would
ha ve been an important comparison since dispersal in blends require s adults to only move short distances away from the refuge plant before having dispersed from their “natal field”. The SAP (2011) stated that “ f or seed mixtures dispersal rates will be 100%

, and greater than between field refuges. The main consequence [of greater dispersal rates] is that seed mixtures will have faster resistance evolution than between field refuges”. BPPD
notes that Pioneer’s dura bility estimates for seed blends would likely be lower than what is currently reported if greater dispersal had been incorporated into the benchmark a n d w o r s t

- c a s e assumptions for SWCB as was done for block refuge simulations.

SAP (2011) recommendations:

**•** Larval moveme nt: explicit vs. implicit Pioneer/DuPont included both the NBI and BNI hypothesis in their model, which addresses the SAP’s (2001) recommendation to include larval movement explicitly and to address pathways of selection before and after Bt exposure. The a pplicant included early instar (varied dominance as well as late instar exposure and mortality
into their model construct.

**•** Cross

- resistance The applicant addressed the SAP’s (2011) and BPPD’s concern about cross

- resistance and incorporated a 10%
and 20% c ross

- resistance for Cry1F to Cry1Ab and Cry1Ab to Cry1F, respectively. Additionally, the applicant
assumed a worst

- case maximum of 30% cross

- resistance for Cry1Ab to Cry1F in their probability analysis. It is
uncertain

, however

, whether enough simulations w ere run to sample extreme values from the parameter range (see discussion in probability analysis above ).- 16

- **•** Non

- uniform oviposition The applicant explored non

- uniform oviposition and its effects on the durability of seed blends in a sensitivity analysis onl y . Their main modeling results were not derived with assumptions of non

- uniform oviposition (both
benchmark and worst

- case simulations). Hence, when looking at Pioneer/DuPont’s estimated durability results in Tables 6 and 7, this point needs to be consider ed. Based on the advice received by the SAP (2011) as well as information published in Hellmich et al. (1999), BPPD concludes that any durability analysis of a seed blend application should conservatively incorporate non

- uniform oviposition favoring Bt pla nts to address situations where greater insect damage to refuge plants in a seed mixture could result in preferring B t p l a n t s a s o v i p o s i t i o n

s i t e s or where CRW protected maize could crowd out refuge plants.

BPPD Conclusions :

Pioneer/DuPont addressed the r ecommendations made in BPPD (2011): the applicant inflated the fecundity of the target pests by 6 and 4

- fold to avoid population extinction for European and southwestern corn borers in their modeling analyses, included some worst

- case values for several se nsitive parameters in their new simulations, conducted a probability analysis to be able to report a risk profile to the Agency, and addressed the most germane uncertainties described by the SAP (2011) for seed blends with pyramided PIPs targeting Lepidopt era. However, Pioneer/DuPont did not include non

- uniform oviposition by Lepidoptera in their main modeling analyses and explore greater female (southwestern corn borer) dispersal out of seed blends.

After careful review of the applicant’s new modeling sub mission, BPPD conclude s that :

**•** The 20% single toxin block refuges had the lowest durability estimates of all options tested in
Pioneer/DuPont’s model

. Those durability results were, however, artificially reduced by higher fecundity assumptions. Pioneer/DuPont estimated blends to be betw een 6 and 60

- fold more du rable than 20% blocks for single toxin PIPs.

**•** T he proposed seed blends (5% and 10%) will likely be substantially less durable than a 5% block refuge
for the same product (100% compliance assumed). This was al so concluded by the SAP (2011). With realistic assumptions of non

- compliance the estimated durability for the block scenario would likely be
somewhat lower but still more durable than the blends. Pioneer/DuPont’s model estimated that a 5% block had close to double the durability of blends for both pests (benchmark assumptions). The durability of the pyramided blends is highly likely to fall in between the two block options. They can be

ex pected to be more durable than the single toxins (especially under assumptions of low cross

r e sistance ). The uncertainties described below ( effects of increased fecundity, no model ing of non

uniform oviposition and greater dispersal out of blends) can be expected to further erode the durability
of blends.

**•** A 10% blend is likely to be more durable t han a 5% seed blend for the same product. Pioneer/DuPont reported, however, that the lower blend percentage had slightly greater durability than the higher blend percentage. Possible explanations for this unexpected outcome are discussed in the uncertainti es section
below.

**•** These conclusions are based on all available information, including Pioneer/DuPont’s modeling, the 2011 SAP report, and EPA/ORD modeling analyses created for other seed blend products. Quantification of the risk (i.e. level of durability) for Optimum AcreMax and Optimum AcreMax Xtra is complicated by uncertainties in some biological parameters including uniform/non

- uniform oviposition, effects of inflated fecundity, degree of female pre

- ovipositional dispersal as well as aspects of the- 17

- pro bability analysis. All these uncertainties together could functionally lower the durability of seed blends relative to block refuges.

**•** In consideration of the cumulative effect of the uncertainties and w itho ut additional modeling , BPPD is
unable to complete ly quantify the relative durabilities of the various refuge options . BPPD expects, however, the relative durability relationship to hold true: 5% pyramid block > 10% pyramid seed blend > 5% pyramid seed blend > 20% single toxin block

.

**•** The 10% Optimum Acre Max 1 seed blend (expressing Cry34/35) targeting corn rootworm was approved in 2009. The applicant seeks to combine this single CRW gene with the pyramid PIP for Lepidoptera into a new 10% seed blend product (Optimum AcreMax Xtra). As stated above, the ris k of
resistance development and relative durability cannot be quantified for any of the proposed blends; however, BPPD expects the risk of resistance evolution to be lower for a 10% seed blend expressing two pyramided PIPs than a 5% blend as proposed for t he Optimum AcreMax product targeting Lepidoptera only.

BPPD has identified the following uncertainties in Pioneer/DuPont’s analysis:

1) Non

- uniform oviposition behavior was not included into Pioneer/DuPont’s benchmark simulations although a sensitivity analysis for this parameter showed a trend of greater durability for the 10% se ed blend over the 5% blend when conservative, yet realistic, assumptions of non

- uniform oviposition favoring Bt plants were included. Based on the advice received by the SAP (2011) as well as information published in Hellmich et al. (1999), BPPD concludes that any durability analysis of a seed blend application targeting lepidoptera should conservatively incorporate non

- uniform oviposition in order to address situations where greater insect damage to refuge plants in a seed mixture could result in
greater o viposition onto Bt plants

. One of Pioneer/DuPont’s proposed products (Optimum AcreMax Xtra) contains a corn rootworm protectant gene; for PIP plants that receive additional below ground protection, early crowding out of refuge plants by Bt plants is anothe r reason why non

- uniform oviposition needs to be addressed in modeling analyses.

2) Pioneer/DuPont’s durability estimates for the 5% seed blend were greater than for the 10% seed blend

s trategy . BPPD determined that this non

- intuitive result may have been a consequence of having inflated the fecundity of the target pests by 6

- and 4

- fold for ECB and SWCB. This unexpected outcome of greater durability for a blend containing a lower non

- Bt proportion was also the topic of discussion in the SAP report (2011) an d Ives et al. (2011). It was demonstrated there that by inflating fecundity greatly that a two

- toxin

- pyramid in a seed blend with low non

- Bt proportions could be driven to have increased durability over a blend with greater non

- Bt proportions when target p ests were mobile . In fact, the SAP demonstrated that at first the durability of seed blends increased with increasing proportion of

non

- Bt plants. Then a precipice was reached were durability began to decrease for blends with greater proportions of non

- Bt plants. As fecundity was successively inflated, the precipice moved towards lower
values of blends with non

- Bt proportions ( Figure 1 ). In other words, when fecundity was greatly increased, the effect was that there was greater mort ality of susceptible genotypes as a result of density

dependence; this led to greater selection of resistant genotypes (Ives et al. 2011). “ Because all sorts of variability are not considered in deterministic simulations, it is possible to push the model o ver the precipice, which marks the point on the graph where populations begin to head towards extinction and the density

- dependent model becomes density

- independent; accelerated resistance evolution can be observed at this point; the precautionary advice i s to stay away from this precipice or boundary ” (Ives, personal communication).- 18

- Since the applicant’s durability estimates appear to be derived from beyond this boundary (precipice), BPPD concluded that it might be more meaningful to look at the sensitiv ity analysis conducted for non

uniform ovi position: when Bt plants were favored as oviposition sites, the fecundity of susceptible
genotypes may have been indirectly reduced (of ECB but not so for SWCB) and density

- dependence
moved Pioneer/DuPont’s model b ack into the realm where durability estimates conformed to expectations under resistance evolution models and where seed blends with greater non

- Bt proportions had greater durability than those with fewer non

- Bt plants . Modeling analyses conducted in the p ast by EPA/ORD also predicted that a greater percentage (i.e. 10%) of refuge plants in a seed blend will be more durable than a lower percentage (i.e. 5%) of refuge plants (BPPD 2010a and BPPD 2010b). BPPD
also considered Carriere et al. (2004) who reporte d that differences in Bt durability predicted by block
and seed blends of equal refuge plant proportion s (caused by a reduction in refuge insects in seed blends) could be overcome by increasing the proportion of non

- Bt plants in seed mixtures.

3) Pioneer/DuP ont conducted a sensitivity analysis for the “number of eggs produced in natal fields” by SWCB in block refuges (single and pyramided PIPs) but not for seed blend scenarios. The applicant reported that durability results for blocks were not very sensitive to changes in this parameter although a slightly decreasing trend in durability was noted for single PIPs as pre

- ovipositional dispersal increased. For a 5% block refuge with a pyramided PIP (where no resistance evolved for the duration of the
simulation

– 400 yrs), the applicant reported that the final resistance allele frequency was slightly higher when there was more dispersal out of the fields. This result agrees with what the SAP (2011) reported, namely, that with high female dispersal, resistance woul d evolve faster. Pioneer/DuPont did not compare the durability of blends to blocks when varying this parameter. This would have been an important relative comparison, since dispersal in blends requires adults to only move short distances away from the
refu ge plant before having dispersed from their “natal field”. Hence the effects of female pre

ovipositional dispersal out of seed blends can be expected to have a greater effect on resistance evolution in seed blends than block refuges. The SAP (2011) stated that “for seed mixtures dispersal rates will be 100%, and greater than between field refuges. The main consequence [of greater dispersal rates] is that seed mixtures will have faster resistance evolution than between field refuges”.

RECOMMENDATIONS :

It is recommended that Pioneer/DuPont incorporate the following into their model: a) Non

- uniform oviposition for both generations of lepidoptera in a 10% seed blend (OAM Xtra); b) Non

- uniform oviposition for the second generation of lepidoptera in a 5% seed blend without a corn rootworm trait; and c) Increased female (SWCB) pre

- ovipositional dispersal out of seed blends.

It is recommended that Pioneer/DuPont further clarify the following about the modeling: d) How was fecundity increased in seed blends? Does the 6

- fold ( and 4

- fold ) increase refer to the fecundity of susceptible insects on refuge plants r e l a t i v e to fecundity on Bt plants? e) How was uniform oviposition dealt with in the model code? The methodology should be explained. Do adults form one big random mating po ol in seed blends

, and eggs are uniformly applied across the field (or else)? f) How was non

- uniform oviposition dealt with in the model code o f t h e s e n s i t i v i t y a n a l y s i s ? Pioneer/DuPont should explain the methodology used.- 19

- BPPD recommends research on: g) Ovipositional behavior in seed ble nds under, for example, increased pest pressure; h) The potential and degree of crowding out refuge plants by Bt protected plants in blends expressing corn rootworm traits under different environmental conditions; and i) Adult movement (related to mating and mov ement 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 res istance (particularly with older instars).- 20

- IV. REFERENCES

Carriere, Y, MS Sisterson, and BE Tabashnik. 2004. Resistance management for sustainable use of Bacillus thuringiensis crops in integrated pest management, p. 65

- 95. Book chapter in “Insect Pest Mana gement: Field and Protected Crops”, Springer, 2004.

Hellmich RL, LS Higgins, JF Witkowski, JE Campbell, and LC Lewis. 1 9 9 9

. Oviposition by European corn borer (Lepidoptera: Crambidae) in response to various transgenic events. Journal of Economic Entomology,

Vol . 92(5): 1014

- 1020

Ives AR, PR Glaum, NL Ziebarth, and DA Andow. 2 0 1 1

. The evolution of resistance to two

- toxin pyramid transgenic crops. Ecological Applications, Vol. 21(2): 503

- 515

AR Ives. 2011, personal ( p h o n e ) communication with J.C. Martinez on August 3 rd , 2011

.

P a n Z

, K T r a n , H Z e n g , a n d o t h e r s ( 2 0 1 1 )

. R e s p o n s e s t o E P A ’ s l e t t e r d a t e d J u n e 7

, 2 0 1 1 r e g a r d i n g P i o n e e r ’ s L e p i d o p t e r a n b l e n d e d r e f u g e p r o d u c t s

. U n p u b l i s h e d s t u d y s u b m i t t e d t o t h e A g e n c y b y P i o n e e r H i

- B r e d

I n t e r n a t i o n a l

, I n c

. ( M R I D 4 8 5 2 1 9

- 0 1 )

.

Other:

BPPD, 2011a. EPA Review of Pioneer’s new product registration request, Optimum AcreMax Insect Protection, expressing Cry1F and Cry1Ab in a 5% seed blend to control lepidopteran pests of corn . Memo from JC Martinez to M. Mendelsohn dated May 18, 2 011.

BPPD, 2011b. Agency deficiency letter sent by Dr. Sheryl Reilly in June, 2011 addressed to Tracy Rood from
Pioneer/DuPont.

BPPD, 2010a. Science Assessment of Pioneer’s modeling addenda in support of their application of registration for Optimum Acr eMax1 Insect Protection. Memo from JC Martinez to M. Mendelsohn dated February 4, 2010.

BPPD, 2010b. Science risk assessment to evaluate the durability of a 10% Cry34/35 seed blend refuge deployment of Cry34/35 corn relative to the durability predicted fo r a 20% block in regards to Pioneer/DuPont’s Optimum AcreMax1. Memo from JC Martinez to M. Mendelsohn dated April 22, 2010.

EPA Scientific Advisory Panel. 2011. Transmittal Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting Held December 8

- 9, 2010 to Address Scientific Issues Associated with Insect Resistance Management for SmartStaxTM Refuge

- in

- the

- Bag, a Plant

- Incorporated Protectant (PIP) Corn Seed Blend. Memorandum from Dr. Sharlene Matten (DFO) to Dr. Steven Bradbury (Director of OPP) on Ma rch 3, 2011.