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, 2010
2

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 rootworm
4

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 pestProposed
7

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 1st 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 2nd, but no against 3rd 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 3rd instars

– P4 = moved to Bt on day 12; 3rd 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 (5th instar, 10%)

SWCB Results: Some survival in P4 (4th instar, 4.2%), high survival 

in P5 (5th instar 80.4%) and P6 (5th instar, 93.3%)

BPPD uncertainties: 

• Propensity for late-instar lepidopteran dispersal

• Whether survival of 5th instars is a function of non-feeding 

• Differences in survival between 5th 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 Estimates
14

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 479437-01
Table 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 Frequencies1 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

1Heterozygote 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 Frequencies1 and Generations to Resistance

Compliant fields Non-Compliant Fields

Cry1A.1051; Cry2Ab21; Cry1F1 Cry1A.1051 Cry2Ab21 Cry1F1

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

1Heterozygote 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 preovipositional 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)

SSa/SSb 19%

RSa/SSb, SSa/RSb 14.5%

RSa/RSb 9.5%

RRa/SSb, SSa/RRb 5%

RRa/RSb, RSa/RRb 5%

RRa/RRb 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 time
26

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

Noncompliant 

Compliant
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 assessment
40

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

D
41

OPPBPPD

!
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 1st 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, 2010
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Section A: Biology of European Corn Borer, 

Southwestern Corn Borer, Corn Earworm, 

and Corn Rootworm
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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?

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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 1st, 2nd, and 3rd instars. But, there was greater 

survivability among 4th and 5th 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. 46
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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. 

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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.

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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.
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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?

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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, densitydependent 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 nonBt plots relative to the surviving larvae in Bt plots.

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Charge Question 5 (continued)

For the SmartStax toxins, Dow/Monsanto made a densitydependent 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 nonadjusted 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. 52
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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.

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Section B: Modeling of Resistance Evolution
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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. 55
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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.

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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? 57
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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); 

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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.

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