## O P P B P P D BPPD’s Preliminary Risk Assessment for a 5% SmartStax Corn Seed Blend Targeting

Lepidopteran and Corn Rootworm Pests

Jeannette Martinez, M.S. & Alan Reynolds, M.S. U.S. EPA, Office of Pesticide Programs Biopesticides and Pollution Prevention Division Washington, DC December 8th , 20102 

### Outline 

## O P P B P P D • Background

– Seed blend concept and history

– SmartStax regulatory history

• New data and biological considerations

– Efficacy studies for SmartStax seed blends

– Larval movement studies

– CRW dose mortality estimates

• Simulation models to evaluate a SmartStax seed blend

– Monsanto deterministic model for ECB and SWCB

– Dow AgroSciences stochastic model for CRW

– EPA/ORD POPGEN model for CRW, ECB, and SWCB

• BPPD conclusions for preliminary risk assessment

## O P P B P P D Seed Blend Concept

• Seed blend technology: refuge seed (non - Bt) and Bt seed are mixed in one bag

• Potential benefits: grower compliance with refuge assured; maturity of refuge and Bt plants synchronized; greater synchrony between adult emergence from Bt and non - Bt plants

• SAP (1998 & 2000): discouraged from using blended concept (Lepidoptera); concern with larval movement, especially survival of heterozygote genotypes

• SAP (2009): Seed blend technology compatible with biology of corn rootworm4 

## O P P B P P D Seed Blend Literature – Some Highlights

• Mallet & Porter (1992)

– Theoretical analysis of seed blends (SB) and structured refuges (SR) simulating mobile pests. Result: SB less durable than SR • Onstad & Gould (1998)

– Modeling resistance evolution in SB and SR for ECB. Result: SB less durable than SR • Davis & Onstad (2000)

– Used field data on larval movement and survival to parameterize Onstad & Gould model. Result: SB less durable than SR • Carriere **et al.** (2004)

– SB not bad ‘per se’; to overcome reduction in effective refuge due to larval movement between Bt and non - Bt plants, need to increase percent refuge seed compared to percent refuge in SR strategy • Onstad **et al.** (2010)

– SB risky for IRM because of larval movement; pest monitoring more challenging. SR are risky due to lower compliance with IRM requirements.

## O P P B P P D SmartStax Regulatory History Registered in 2009 by Monsanto and Dow • Five toxin pyramided stack targeting lepidoptera (ECB, SWCB, CEW) and corn rootworm pests (WCR, NCR)

• Derived from MON 89034 (Cry1A.105, Cry2Ab2) x Herculex XTRA (Cry1F, Cry34/35) x MON 88017 (Cry3Bb1) Current refuge: 5% structured refuge for both pest complexes (Corn Belt only) • Separate refuges

– For CRW: infield strips or adjacent

– For lepidoptera : up to 1⁄2 mile away from Bt field • Common refuge

– adjacent non

- Bt Lep. refuge Active Toxins Corn rootworm Cry34/35Ab1 Cry3Bb1 Lepidoptera Cry1A.105 Cry2Ab2 Cry1F

## O P P B P P D New Proposal by Monsanto and Dow 5% SmartStax (SSX) seed blend for both pest complexes • 5% of seed express non - Bt (red plants) • 95% of seed express Bt (green plants)

• 5% SSX seed blend proposed for Corn Belt and some regions of Cotton Belt where corn earworm (CEW) not a major pestProposed7 

### Outline 

## O P P B P P D • Background

– Seed blend concept and history

– SmartStax regulatory history

• New data and biological considerations

– Efficacy studies for SmartStax seed blends

– Larval movement studies

– CRW dose mortality estimates

• Simulation models to evaluate a SmartStax seed blend

– Monsanto deterministic model for ECB and SWCB

– Dow AgroSciences stochastic model for CRW

– EPA/ORD POPGEN model for CRW, ECB, and SWCB

• BPPD conclusions for preliminary risk assessment

## O P P B P P D Monsanto/Dow Field Efficacy Studies a. Field studies for 5% SSX seed blend (SB) targeting main pests; Monsanto/Dow concluded “high efficacy” with “little to no damage”, “no to few survivors” on SSX plants: • ECB  BPPD concurs • SWCB  BPPD concurs; SSX was somewhat less efficacious than ECB based on damage and survival results • CEW  BPPD concurs; under high - infestation and greater pest pressure, however, SSX is somewhat less efficacious • CRW  BPPD concludes SSX has good efficacy against pest; SSX is less efficacious against older larvae BPPD uncertainties: • Additional protection for non - Bt plants was observed in seed blends; concern with impacts on effective refuge (ER) in SB • Male CRW emergence was variable and reduced up to 60 times in 5% seed blends  concerns with effects on durability of seed blend

## O P P B P P D Monsanto/Dow Larval Movement Studies b. Field and lab larval movement studies in 5% SSX SB, survival of different larval stages on Bt, and Monsanto and Dow conclusions: • ECB and SWCB: reduced ER in SSX SB possible; complete control of 1 st to 4th instars. No larval movement expected  BPPD: some survival possible for 4th and 5th instars based on lab results • CRW: SSX SB high efficacy against neonates, significant efficacy against 2 nd , but no against 3 rd instar. “Little or no selection expected” because of tolerance to Bt toxins  BPPD concurs on mortality conclusion yet questions ‘no concern for selection’ because of Bt tolerance BPPD uncertainties: • Propensity for lepidoptera late - instar larval movement • Characterizing/quantifying the tolerance of CRW to Cry34/35Ab1 and Cry3Bb1 and implications for selection

## O P P B P P D Monsanto Larval Movement Study Monsanto lab experiment simulating larval movement:

• Different exposure treatments for seven distinct populations (50 larvae/each)

• Populations and simulated movement onto Bt

– P1 = remained on non - Bt; negative control

– P2 = moved to Bt on day 4; second instars

– P3 = moved to Bt on day 8; mostly 3 rd instars

– P4 = moved to Bt on day 12; 3 rd and 4th instars (ECB); 4th instars (SWCB)

– P5 = moved to Bt on day 16; 4th and 5th instar (ECB); mostly 5th instars (SWCB)

– P6 = moved to Bt on day 20; 5th instars

– P7 = on Bt for the duration of the experiment; positive control
• Duration of experiment 29 - d total for ECB, 26 -d for SWCB • Mortality measured for each larval population (intervals, final)

## O P P B P P D Monsanto Larval Movement Study ECB Results : High control mortality in P1; some survival in P5 (4th and 5th instar, 6%) and P6 (5 th instar, 10%) SWCB Results : Some survival in P4 (4th instar, 4.2%), high survival in P5 (5th instar 80.4%) and P6 (5 th instar, 93.3%)

BPPD uncertainties: • Propensity for late - instar lepidopteran dispersal • Whether survival of 5 th instars is a function of non -feeding • Differences in survival between 5 th instar SWCB vs. ECB Monsanto’s final results were not adjusted for control mortality (MRID 479437 -01, Appendix 1) Target

Pest Final % Survival P1 P2 P3 P4 P5 P6 P7 ECB 42.6 0 0 0 6.4 9.4 0 SWCB 85.7 0 0 4.2 80.4 93.3 0

## O P P B P P D Corn Rootworm - Dose Mortality Estimates CRW

– Below ground pest; challenging to get dose mortality estimates

– Methodology for dose estimates developed (Storer et al. 2006 and others at USDA/ARS)

– Two different studies on how to handle densitydependence (Onstad et al. 2006; Hibbard et al. 2010) Dow/Monsanto: Density-dependent adjustments to dose estimates for the SSX toxins based on egg density/survival relationships as discussed by Onstad et al. (2006). Artificial infestations occurred with 500 eggs/plant (approx. 750 eggs/row foot). Target Pest Cry3Bb1 Cry34/35Ab1 Pyramid % Dose mortality estimate % Dose mortality estimate % Dose mortality estimate Corn rootworm 99.75 99.75 99.95

## O P P B P P D Hibbard et al. (2010) found density- dependent mortality not triggered below 851 eggs/foot (multi -year data analysis)  Density-dependent threshold higher than at artificial infestation level tested by Monsanto/Dow. Net impact: dose estimates are lower for CRW when Hibbard et al. ’s method is used. BPPD considered both methods; recommended in 2009 risk assessment of SSX that dose should also be evaluated without a density-dependent adjustment. The non - adjusted dose estimates for SSX toxin are:

BPPD uncertainty • Which dose estimates are more appropriate given the different thresholds for density-dependent effects? Target Pest Cry34/35Ab1 Cry3Bb1 Pyramid % Dose mortality % Dose mortality % Dose mortality Corn rootworm 94.2 97.5 98.2 

### Corn Rootworm Dose Mortality Estimates14 

### Outline 

## O P P B P P D • Background

– Seed blend concept and history

– SmartStax regulatory history

• New data and biological considerations

– Efficacy studies for SmartStax seed blends

– Larval movement studies

– CRW dose mortality estimates

• Simulation models to evaluate a SmartStax seed blend

– Monsanto deterministic model for ECB and SWCB

– Dow AgroSciences stochastic model for CRW

– EPA/ORD POPGEN model for CRW, ECB, and SWCB

• BPPD conclusions for preliminary risk assessment

## O P P B P P D Monsanto Modeling Analysis: ECB and SWCB Model structure (ECB/SWCB):

– Three locus deterministic spatially implicit model

– Model run time: 100 generations or until resistance evolved (i.e. RAF = 0.5) Assumptions:

– No cost to resistance

– Non - compliance for SR: random vs. non - random (0%, 20%, and 40%)

– Seed blend modeled as 5% structured refuge w/ 1%, 2.5%, and 5% (ER) (field data support 3x reduction in ER)

– Fitness of heterozygotes 2x and 5x susceptible genotype

– Mixing between compliant and non - compliant compartments varied

– Non - random oviposition behavior in fields

## O P P B P P D Monsanto Modeling Analysis: ECB and SWCB Parameter values:

– IRAF for Cry1A.105, Cry2Ab2, Cry1F = 0.005

– Mixing between compartments: 0%, 50%, and 100%

– Dose of toxins (two different scenarios)
Dose assumption Percent Dose Mortality Toxin 1 Toxin 2 Toxin 3 ECB benchmark 99.9% 99.9% 99.9% ECB worst- case 99% 99% 99% SWCB benchmark 99% 90% 99% SWCB worst- case 99% 85% 99% Modified from Monsanto submission Appendix 8 (Table 1), MRID 479437-01Table 34 . Number of generations to ECB resistance and final R-allele frequency with worst-case assumptions and different scenarios for 5% seed mixtures and structured refuge% Refuge%Non-compliance% Mixing Final Frequencies 1 and Generations to Resistance: Compliant fields Non - Compliant Fields Cry1A.105; Cry2Ab2; Cry1F Cry1A.105; Cry2Ab2; Cry1F 1 0 0 0.005; >100 2.5 0 0 0.005; >100

5 0 0 0.005; >100 5 20 0 0.005; >100 0.69; 3 20 50 0.005; >100 0.03; >100 20 100 0.005; >100 0.03; >100 40 0 0.005; >100 0.69; 3 40 50 0.005; >100 0.03; >100 40 100 0.005; >100 0.03; >100 Table modified from Appendix 8, p.644, MRID 479437-01 1 Heterozygote fitness is 5x that of the susceptible genotype 1%, 2.5%, and 5% refuges with 0% non - compliance represent seed mixture scenarios Bolded lines show worst-case scenarios and results or seed mixtures and results 

## O P P B P P D Monsanto Modeling – ECB Results No ECB resistance within timeframe modeled for worst- case assumptions in seed blends (simulations stopped at 100 gen.)Table 36 . Number of generations to SWCB resistance and final R-allele frequency with worst-case assumptions and different scenarios for 5% seed mixtures and structured refuge% Refuge%Non-compliance% Mixing Final Frequencies 1 and Generations to Resistance Compliant fields Non - Compliant Fields Cry1A.105 1 ; Cry2Ab2 1 ; Cry1F 1 Cry1A.105 1 Cry2Ab2 1 Cry1F 1 1 0 0 0.011; >100 2.5 0 0 0.007; >100

5 0 0 0.006; >100
5 20 0 0.006; >100 0.69; 3 0.512; 4 0.69; 3 20 50 0.006; >100 0.031; >100 0.027; >100 0.031; >100 20 100 0.006; >100 0.032; >100 0.028; >100 0.032; >100 40 0 0.006; >100 0.69; 3 0.512; 4 0.69; 3 40 50 0.006; >100 0.031; >100 0.027; >100 0.031; >100 40 100 0.006; >100 0.034; >100 0.029; >100 0.034; >100 Table modified from Appendix 8, p.640, MRID 479437-01 1 Heterozygote fitness is 5x that of the susceptible genotype 1%, 2.5%, and 5% refuges with 0% non - compliance represent seed mixture scenarios Bolded lines show worst-case scenarios and results or seed mixtures and results 

## O P P B P P D Monsanto Modeling – SWCB Results No SWCB resistance within timeframe modeled for worst- case assumptions in seed blends (simulations stopped at 100 gen.)

## O P P B P P D BPPD Identified Uncertainties for Lepidoptera Modeling Seed blend technology :

– Standard error of mixing not communicated and incorporated into model (assumption there is no variability) ECB:

– Local vs. long distance dispersal: assumption of predominantly local dispersal; yet low recapture rates from mark- recapture studies (<1%)

– Long -distance dispersal observed and reported: frequency?

– Is a 5% SSX SB a durable IRM strategy with long distance adult movement?

– Is there female pre - mating and/or pre - ovipositional dispersal

## O P P B P P D BPPD Identified Uncertainties for Lepidoptera Modeling SWCB:

– ECB typically used as surrogate sp; realistic for larval and adult movement of SWCB?

– Is there long - distance, female pre - mating, and/or pre ovipositional dispersal?

– Some evidence for greater (natal) field fidelity in SWCB than for ECB. If mating primarily in natal fields, then seed mixtures potentially preferred IRM strategy

## O P P B P P D Dow Modeling Analysis - CRW Model structure:

– Stochastic, spatially explicit (adapted from Storer 2008)

– Landscape consisted of 10 x 10 grid fields, each 25 ha in size

– Model run for 20 generations; each simulation run 5x to determine role of stochastic processes on resistance evolution • SR with 0% non - compliance, variable non - compliance • 5% SB with different parameter assumptions

## O P P B P P D Dow Modeling Analysis - CRW Assumptions:

– Cross - resistance between two loci negligible

– Initial RAF = 0.005

– Benchmark dose estimates based on DD adjustment (as presented earlier)

– Heterozygote genotype had intermediate fitness between susceptible and resistant genotypes

– 7-day developmental delay for susceptible genotype on Bt

– Complete resistance (no developmental delay for heterozygotes); no cost to resistance

– No female pre - mating dispersal

– Adult male movement 6 rows/day

– Willful non - compliance (20%; fixed fields each year) and accidental non - compliance with refuge plantings (16%; random placement)

## O P P B P P D Dow Modeling Analysis - CRW Parameter values:

– Fecundity of females increased to 100 eggs/female to avoid population extinction

– Different probabilities for adults leaving Bt or refuge row

– Benchmark dose Cry34/35, Cry3Bb1, and pyramid; also low dose (single traits 99%, pyramid 99.9%) and worst case dose scenarios (BPPD recommended)

– Genotype specific probability of leaving a Bt plant Genotype Prob (leaving Bt Plant) SS a/SS b 19% RS a/SS b , SS a/RS b 14.5% RS a/RS b 9.5% RR a/SS b , SS a/RR b 5% RR a/RSb, RSa/RR b 5% RR a/RR b 0%

## O P P B P P D Dow CRW Modeling - Results Overall : • Rate of increase of RAF similar for seed blends and structured refuge with benchmark dose, low dose, larval movement, and survival consistent with empirical observations Rate of Adaptation in Seed Blend Rate of Adaptation in Structured Refuge Benchmark dose & Dow’s low dose Increase in RAF similar; less than two-fold increase in 20 yrs BPPD recommended dose Increase in RAF but lower than for SR Rate doubled in 7 gen Rate of adaptation here means ‘increase in resistance allele frequency over timeframe modeled’

## O P P B P P D BPPD Identified Uncertainties for CRW Modeling

• Seed blend technology: standard error of mixing Bt and non - Bt seed not communicated and incorporated into model • Is initial resistance allele frequency too low? No sensitivity analysis provided for this parameter • Actual values of final RAF not provided by Dow (graphical output). Unclear which differences between scenarios were significant (focus on SB scenarios supported by empirical data vs. structured refuge) • Long -distance, pre - ovipositional dispersal not incorporated in model although supported by some studies in literature. Shown to increase durability in block refuges (Caprio & Glaser 2010) • Reduced male emergence in 5% seed blend not incorporated into analysis. Effect on durability unknown at this time26 

## O P P B P P D EPA/OPP/BPPD and EPA/ORD Working Relationship

• Recently developed EPA/ORD model, POPGEN, for use in IRM risk assessments.

• Authors are: • Dr. John Glaser, EPA/ORD, National Risk Management Research Lab, Cincinnati, OH • Dr. Mike Caprio, Mississippi State University, MS, contractor to the EPA

• EPA/ORD collaborated with BPPD to conduct an independent modeling analyses for lepidoptera and corn rootworm

• BPPD recommended incorporation of some biological aspects of target pests and values for parameters of uncertainty

## O P P B P P D EPA/ORD Modeling Analysis

• POPGEN

– Frequency based, two locus deterministic, probabilistic model for CRW

– Frequency based, three locus deterministic, probabilistic adaptation of CRW version for lepidoptera analysis

– Probabilistic analysis used PERT-distribution for parameters of uncertainty (min, mode, max)

• Main objective :

– Compare durability between 5% seed blend and 5% structured refuge strategy

– Estimate probability of occurrence for predicted durability results

## O P P B P P D EPA/ORD Modeling Analysis Model structure for CRW and lepidoptera :

– Two compartment model • persistent non - compliance (fixed fields) • compliant fields and accidental non - compliance

– Model run as a) seed mixture or b) structured refuge

– Each version run for 1000 (CRW) and 2000 simulations (lepidoptera), each time re -sampling parameters of uncertainty and recording time to resistance

– Results were summarized in density distribution Non compliantCompliant

## O P P B P P D EPA/ORD Modeling Analysis – CRW Assumptions:

– No cross - resistance between two loci

– No refuge for non - compliant fields

– ORD’s effective refuge size (SR) = “prop. of eggs in a field that start out underneath a new refuge” at the beginning of each simulation • non - random oviposition • adult dispersal • refuge placement (random vs. fixed): in ER • female pre - ovipositional dispersal  in ER

– Asymmetrical larval movement from Bt  non - Bt plant

## O P P B P P D EPA/ORD CRW Modeling – Probability Analysis Uncertainty distribution (PERT- beta):

• Beta distribution models events constrained by intervals •Tool to identify risk based on expert estimates (min, mode, max); some information about parameter value is known (not completely random) • Designed to generate distribution similar to normal curve

•Values near the mode of the distribution are more likely than others

## O P P B P P D EPA/ORD Modeling Analysis - CRW Assumptions for parameters of uncertainty: PERT- beta distribution (mode weighted by 4) for following parameters:

... and other parameters not shown here Sensitivity analyses: ANOVA, step -wise regression, standardized regression, Bayesian Model analysis (* - denotes sensitive parameters) Parameter Minimum Mode Maximum Initial RAF* 0.001 0.005 0.02 Overall survival* 0.0007 0.018 0.03 Dominance* 0.01 0.05 0.2 Structured refuge* 0 0.03 0.06 Non - compliance* 0 0.3 0.6 Non - random mating 0.2 0.5 0.7

## O P P B P P D EPA/ORD Modeling – CRW Results Distribution of results from 1000 simulation (shown for first 200 generations): SM durability > SR durability

## O P P B P P D EPA/ORD Model – CRW Results

Quantile/ Probability Seed blend Struct. Ref. 0% 23 yrs 12 yrs 5% 33 yrs 22 yrs 10% 39 yrs 26 yrs 25% 51 yrs 34 yrs 50% 70 yrs 45 yrs

## O P P B P P D EPA/ORD Modeling – CRW Results

Quantile/ Probability Seed blend Struct. Ref. 0% 23 yrs 12 yrs 5% 33 yrs 22 yrs 10% 39 yrs 26 yrs 25% 51 yrs 34 yrs 50% 70 yrs 45 yrs

## O P P B P P D BPPD Risk Assessment – CRW Results A general qualitative (non - quantitative) comparison between EPA and Dow results shows: Despite differences in models, Dow and EPA/ORD predicted similar outcomes for a 5% SmartStax SB and SR; difference observed (EPA/ORD) statistically significant at 5% level of risk

BPPD uncertainties:

– BPPD assumed high non - compliance ( i.e., mode and max)

– Margin of error (+/-) for seed blend refuge not considered Refuge Scenario Dow CRW Model EPA/ORD CRW model Increase in Resistance Allele Frequency 5% Level of Risk Mean Time to Resistance 5% Seed Blend Similar rates of increase for the 20 years modeled 33 years 81.9 years 5% Structured Refuge 22 years 52.9 years

### EPA/ORD Lepidoptera Model – Assumptions

• No cross - resistance between three loci • Probability distribution addressing uncertainties for different pests

ECB Parameter Cry1A.105 (mode) Toxin Cry2Ab2 (mode) Cry1F (mode) Fitness 0.001 (0.05 - SWCB) 0.001 (0.05 - SWCB) 0.005 (0.005 - SWCB) ECB Parameters Minimum Mode Maximum IRAF* 0.001 0.005 0.02 Dominance* 0.01 0.05 0.2 Str. ref. Size* 0.025 0.04 0.06 Non - compliance* 0 0.3 0.6 Base move.* 0.3 0.5 0.7 Asymm move. 0.8 0.9 0.95 Days of selection* 5 10 15 

## O P P B P P D

* denotes sensitive parameters

## O P P B P P D BPPD Risk Assessment – ECB Results

• SmartStax seed blend 7 times less durable than structured refuge; differences observed (EPA/ORD) statistically significant • Seed blend still an overall durable IRM strategy

BPPD uncertainties:

– Whether predicted durability of SR in EPA’s model is realistic

– How non - multiplicative interactions for >2 loci should be partitioned Refuge Scenario Monsanto ECB Model EPA/ORD ECB model Time to Resistance Level of Risk Time to Resistance 5% Seed Blend >100 gen (>33 or >50 yrs) 0% 158 gen. (52 yrs or 79 yrs) 5% 451 gen (150 yrs or 225 yrs) 5% Structured Refuge >100 gen (>33 or >50 yrs) 0% 1125 gen (375 yrs or 562 yrs) 5% 3055 gen (1018 yrs or 1527 yrs)

## O P P B P P D BPPD Risk Assessment – SWCB Results

• SmartStax seed blend 1.6 times less durable than structured refuge; difference observed is not statistically significant at 5% • Seed blend still estimated to be overall durable IRM strategy

BPPD uncertainties:

– SWCB fitness on SSX higher than for ECB (i.e., mode)

– Due to lack of data, same larval dispersal assumptions were used for both pests; more conservative assumptions made with respect to random mating Refuge Scenario Monsanto SWCB Model EPA/ORD SWCB Model Time to Resistance Level of Risk Time to Resistance 5% Seed Blend >100 gen (>33 or >50 yrs) 0% 73 gen (24 yrs or 36 yrs) 5% 138 gen (46 yrs or 69 yrs) 5% Structured Refuge >100 gen (>33 or >50 yrs) 0% 118 gen (39 yrs or 58 yrs) 5% 207 gen (69 yrs or 103 yrs)39 

### Outline 

## O P P B P P D • Background

– Seed blend concept and history

– SmartStax regulatory history

• New data and biological considerations

– Efficacy studies for SmartStax seed blends

– Larval movement studies

– CRW dose mortality estimates

• Simulation models to evaluate a SmartStax seed blend

– Monsanto deterministic model for ECB and SWCB

– Dow AgroSciences stochastic model for CRW

– EPA/ORD POPGEN model for CRW, ECB, and SWCB

• BPPD conclusions for preliminary risk assessment40 

### BPPD Risk Assessment – Overall Conclusions Two analyses each for each pest complex with different model structures, assumptions, parameter values, sensitivity analyses:  all estimate high durability for 5% SSX seed blends

• BPPD conclusions:

– For CRW: 5% SSX SB high durability • Dow: SB as durable as 5% SR (at worst) • EPA/ORD: SB more durable than 5% SR (1.5 fold)

– For ECB: 5% SSX SB high durability • Monsanto: SB as durable as structured refuge within 100 gen • EPA/ORD: SB 7 times less durable than 5% SR (at worst)

– For SWCB: 5% SSX SB high durability • Monsanto and EPA/ORD: SB as durable as structured refuge 

## O P P B P P D41 

## O P P B P P D 

# !42 

## O P P B P P D A schematic diagram following a cohort of 500 CRW larvae in an experiment to estimate dose

Bt Field 500 larvae per foot Non - Bt Field 500 larvae per foot 99.9% Bt mortality 0.5 larvae left
No Bt mortality 500 larvae left Eggs 1 st instar larvae Older larvae No densitydependent mortality 0.5 larvae left
98% densitydependent mortality 10 larvae left 0.5 adults emerge

10 adults emerge Adults

Reproduced from Figure 1 in Monsanto/Dow’s April 9, 2009 submission (No MRID number)43 

## Corn Seed Blend Proposal Containing Refuge Seed for Lepidopteran and Corn Rootworm Target Pests: EPA Overview and IRM Considerations 

# Charge to the Panel
**Date**: December 8 and 9, 201044 

## Section A : Biology of European Corn Borer, Southwestern Corn Borer, Corn Earworm, and Corn Rootworm45 

## Charge Question 1 European corn borer (ECB) has both local and long distance dispersal capability. Currently, the proportion and frequency of individuals in a population engaging in dispersal before or after mating is unclear. While it has been established that ECB mate in aggregation sites near cornfields, mark - release - recapture studies in the U.S. have typically had a low recapture success (<1%). Recently it was suggested that 1 - day old female ECB may engage in obligate pre - mating dispersal (Dorhout et al. 2008). Please comment on the uncertainties regarding ECB movement including mating sites, pre - mating dispersal, and the proportion of the population engaging in long - distance dispersal. How might these aspects of ECB movement affect a potential seed blend strategy? 4546 

## Charge Question 2 Scientific Advisory Panels (1998 and 2000) discouraged the Agency from the use of Bt seed mixtures to control lepidopteran target pests because substantial larval movement could be expected between Bt and non - Bt plants which could lead to more rapid selection of resistance. BPPD has reviewed new data developed by Dow and Monsanto simulating the effects of SmartStax on various instars of potentially mobile lepidoptera. These data provide evidence that SmartStax is highly toxic to 1 st, 2 nd , and 3 rd instars. But, there was greater survivability among 4 th and 5 th instars. While it has been established that ECB disperse as neonates, there is some uncertainty with respect to lepidopteran propensity for dispersal off non - Bt plants as later instars. BPPD notes that simulation models incorporating data on high larval mortality on SmartStax plants have (in some cases) predicted that seed blends may be as durable as structured refuges. 4647 

## Charge Question 2 (continued)
Please comment on ECB larval plant - to - plant movement including uncertainties about late - instar movement and the potential effect on the durability of a seed blend strategy.

4748 

## Charge Question 3 It is typically assumed that, since European corn borer (ECB) and southwestern corn borer (SWCB) are similar in many ways, ECB can serve as a surrogate for SWCB to address uncertainties regarding biology and genetics. The applicants’ efficacy data, however, suggest that SmartStax is somewhat less toxic to older instars of SWCB. Results of a larval exposure study by Monsanto showed that SWCB survival was higher than ECB on SmartStax. Should some SWCB larvae disperse as older instars, the rate of adaptation to SmartStax could increase in a seed blend deployment. BPPD concluded that simulation models should incorporate such information in their analyses. There is currently a lack of data on the propensity of SWCB larval plant- to plant movement and on how ECB and SWCB differ in this respect, if at all.

4849 

## Charge Question 3 (continued) Please comment on the assumption that ECB is a suitable biological surrogate for SWCB and BPPD’s concerns that a SmartStax seed blend may affect SWCB differently than ECB.50 

## Charge Question 4 Corn earworm (CEW) was not considered in the applicants’ and EPA/ORD’s analyses for a 5% SmartStax seed blend based on the assumption that the insect does not overwinter in the Corn Belt where the blend has been proposed. BPPD is concerned, however, that there could be areas in the southern portion of the Corn Belt where CEW may be able to successfully overwinter, particularly in less severe winters. Such areas may need to be identified because they could contribute to increased selection for CEW resistance to Bt corn (including the proposed 5% SmartStax seed blend). Please comment on the assumption that corn earworm does not successfully overwinter in the Corn Belt and poses less of a risk for resistance. If CEW can potentially overwinter in parts of the Corn Belt, should the insect be considered in the analysis of the proposed 5% SmartStax seed blend? 5051 

## Charge Question 5 To assess dose expression for corn rootworm (CRW) Bt toxins, the level of survival (adult emergence) is typically compared between artificially infested Bt and non - Bt corn plots. However, density dependent mortality in non - Bt plots can potentially confound the comparison by reducing overall survival and adult emergence. (Density
- dependent mortality is not expected in Bt plots due to effects of the toxin on young larvae.) To account for this effect, the dose calculation can be adjusted by removing density - dependent mortality from the control plots. This effectively increases the dose mortality estimate by raising the number of larvae present in non Bt plots relative to the surviving larvae in Bt plots.

5152 

## Charge Question 5 (continued) For the SmartStax toxins, Dow/Monsanto made a density
dependent adjustment to their dose estimates based on density/survival relationships developed by Onstad et al. (2006). The resulting dose mortality profile was: Cry34/35Ab1 (99.75%), Cry3Bb1 (99.75%), and Cry34/35Ab1/Cry3Bb1 pyramid (99.95%). On the other hand, BPPD has also considered separate work by Hibbard et al. (2010), which suggests that density
- dependent mortality occurs at higher egg density levels than those assumed by Dow/Monsanto. In light of this research, BPPD recommended in its 2009 risk assessment of SmartStax that dose should also be evaluated without a density - dependent adjustment. The non adjusted dose profile for the SmartStax toxin is: Cry34/35Ab1 (94.2%), Cry3Bb1 (97.5%), and Cry34/35Ab1/Cry3Bb1 pyramid (98.2%). Please comment on dose estimates for the SmartStax toxins (Cry34/35Ab1 and Cry3Bb1) targeting corn rootworm given the different interpretations of density- dependent mortality. 5253 

## Charge Question 6

Northern and western corn rootworm studies have shown that male emergence in 5% seed blends can be variable and may be up to 60 times lower compared to emergence in non - Bt plots (Data submitted by Monsanto). This information was not included in any of the models used in the SmartStax seed blend analysis. The SAP (2009) concluded that a reduction in the number of males from Bt seed blends could have a negative impact on the effective refuge. BPPD is concerned with the potentially negative effects a reduction in male emergence might have on product durability. Please comment on the potential effects of lowered male emergence of Northern and Western corn rootworm on the durability of the seed blend and whether this information should be incorporated into the risk assessment. 5354 

## Section B : Modeling of Resistance Evolution55 

## Charge Question 7 The durability of the proposed 5% SmartStax seed blend strategy was compared to the durability of a 5% structured refuge for lepidoptera and corn rootworm target pests. Monsanto developed a deterministic three locus model for ECB/SWCB and Dow created a stochastic two locus model for CRW. Separate analyses were conducted using EPA/ORD’s two locus and three locus deterministic, probabilistic model to estimate the risk of resistance evolution with a 5% seed blend and structured refuge. The applicants and EPA/ORD each made conservative assumptions, though of differing degrees, for parameters determined to be sensitive in the models. For example, more conservative initial resistance allele frequencies and fitness assumptions significantly lowered the time to resistance in EPA/ORD’s model for ECB and SWCB. In Monsanto’s modeling of ECB and SWCB, a greater degree of dispersal between compliant and non - compliant fields significantly affected the estimated time to resistance. 5556 

## Charge Question 7 (continued) Please comment on the appropriateness of the assumptions and inputs used for the following parameters in the Monsanto, Dow, and EPA/ORD models: 1. Initial resistance allele frequency for single traits Cry1A.105, Cry2Ab2, Cry1F, Cry34/35Ab1, and Cry3Bb1 for all modeled pests; 2. Survival/fitness for all modeled pests; 3. Dispersal for ECB and SWCB as modeled by Monsanto and EPA/ORD.

5657 

## Charge Question 8 EPA/ORD encountered challenges in the lepidopteran modeling with partitioning non - multiplicative interactions that occurred between more than two resistance genes since the mortality caused by each locus was not independent. With two gene pyramids this non - additivity can be assigned to the single two locus interaction, but in a three gene pyramid there are three possible two locus interactions. In the absence of data, this non - additivity was partitioned equally among the three two locus interactions. As more than two Bt genes are pyramided, this problem will have to be addressed so that resistance evolution in the target pests to these products can be more accurately simulated. Does the Panel have any recommendations for distributing non - multiplicative interactions in models to evaluate multi - gene pyramided products? 5758 

## Charge Question 9

Based on a review of the submitted simulation modeling, the preliminary conclusions are:

• For CRW, a 5% seed mixture and a 5% structured refuge had comparable durability in both the EPA and Dow models; • For ECB , a 5% seed mixture was less durable than a 5% structured refuge in simulations with EPA’s model. However, ECB resistance did not evolve within 158 generations in any of the simulations with the 5% seed mixture, similar to the level of durability predicted by Monsanto’s model. There was no difference in durability between the 5% seed mixture and the 5% structured refuge in Monsanto’s model. Resistance did not evolve to either refuge option within 100 generations (the extent to which the model was run); 5859 

## Charge Question 9 (continued)

• For SWCB , a 5% seed mixture was less durable (78 generations) than a 5% structured refuge (118 generations) in EPA’s model simulations. Conversely, with Monsanto’s model there was no difference in the prediction for durability between the 5% seed mixture and the 5% structured refuge. Resistance did not evolve to either refuge option within 100 generations (the limit of the model simulations). Please comment on the reliability of the estimates of resistance evolution by each of the three models in light of the biological and parameter uncertainties identified by BPPD. 59