UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE 01- CHF\1lCAl. SAl ETY 

Al"D POLL! TION PRE\TNT!ON 

MEMORANDUM 

SUBJECT: 

TO: 

FROM: 

PEER 

REVIEW: 

Review of Com Rootworm Resistance Monitoring Submissions and 

Data for Bt com products containing mCry3A. 

EPA Reg Nos.: 67979-5, -8, -13, -17, -20, -23, -24; 29964-13, -14, -15. 

MRID#s: 492065-01, 493572-01, -02. 

PC Code: 006509. 

Decision#: 483482. 

DP Barcodes: 415366, 420081. 

Ann Sibold, Regulatory Action Leader 

Microbial Pesticides Branch 

Biopesticides and Pollution Prevention,Divisi (7511P) ·,'' / ' 

Alan Reynolds, Entomologist {A_ 1 

Microbial Pesticides Branch .._,, 

Biopesticides and Pollution Prevention Division (7511P) 

Jeannette 

Microbial 

Martinez, 

Pesticides 

Ph.D., 

Branch 

Ecologist/Entomologist ~ 

Biopesticides and Pollution Prevention Division (751 

Action Requested 

The BPPD IRM Team1 has been asked to review com rootworm resistance monitoring data and 

information regarding the mCry3A Bt com toxin. Submissions were made by Syngenta Seeds, 

Inc. (hereafter Syngenta) to .address terms of registration for Bt com products that express 

mCry3A in event MIR604, including the Agrisure RW product line. These reports include 

annual resistance monitoring data (using diet bioassays) for the 2012 growing season and 

unexpected damage reports from the 2011 and 2012 seasons. In addition, this review considers 

reports from independent researchers related to potential cross resistance between mCry3A and 

Cry3Bbl. 

1 The BPPD lRM Team consists of Alan Reynolds and Jeannette Martinez 
Conclusions and Recommendations 

1) Diet bioassays (i.e., growth inhibition, ECso) generally showed that populations collected in 

2012 were sensitive to mCry3A and responded similarly to CRW collected from previous 

seasons, with the exception of one population (Floyd County, IA; see #2 below). The results 

from mortality-based testing (LCso) were also consistent among the 2012 populations, though 

more variable than the ECso assays. 

2) One population collected in 2012 (Floyd County, IA) had approximately 6 to 10 fold higher 

growth inhibition (ECso = 7.05 J.!g/cm2

) and lethality (LCso = 65.20 j.!g/cm2

) than the other tested 

populations (ECso range = 0.03 - 1.45 J.!g/cm2

; LCso range = 1.19 - 8.16 J.!g/cm2

). Syngenta 

characterized this population as indicative of a potential shift in susceptibility to the mCry3A 

toxin. However, the company did not designate the population as "resistant," though the 

resistance ratio (relative to the control population) was 58.2. Syngenta indicated that the area 

would be intensively sampled in subsequent years; presumably this was performed during the 

2013 growing season (data from 2013 had not yet been submitted at the time ofthis review). It 

was not clear from the report whether any additional investigation was planned, or if the 

population was maintained for further study. 

The Floyd County population highlights the BPPD IRM Team's ongoing concern with the use of 

diet bioassays to make resistance determinations. The population is clearly significantly less 

susceptible to mCry3A than any other field population tested since 2008. Yet, it is not clear 

what level of response would be needed to deem the population as mCry3A-resistant. For future 

testing, the BPPD IRM Team recommends that field-collected populations should be tested with 

an on-plant assay (e.g., Gassmann et al. 2011 , Nowatzki et al. 2008) to buttress resistance 

determinations. 

3) In 201 1, Syngenta reported that 12 CRW populations were collected from fields with 

unexpected damage, eight of which were viable for testing. Bioassays showed that these 

populations appeared to still be susceptible to mCry3A as measured by ECsos in diet bioassays. 

Syngenta attributed the damage to high pest pressure or environmental factors; the BPPD IRM 

Team believes these are reasonable explanations for the damage. Three damaged fields were 

also investigated during the 2012 growing season, two of which produced sufficient eggs for 

bioassays. Both of these populations had ECso values comparable to other tested populations, 

though one (Rock Valley, IA) had a LCso value that was almost 38 fold higher than the 

susceptible control colony. Syngenta characterized these results as being mixed, while noting 

that the Rock Valley population exhibited "a degree of tolerance" to the toxin. The BPPD IRM 

Team is concerned that this population (and other populations with similar bioassay responses) 

could be resistant to mCry3A and recommends that in the future all such populations also be 

tested with' on-plant assays. 

4) Work by Dr. Aaron Gassmann (Iowa State University) has documented field-evolved 

resistance to mCry3A (Gassmann et al. 2014). Three CRW populations were collected from 

severely damaged mCry3A fields in northern Iowa during the 2011 season Otis unclear whether 

the damage sites were also investigated by Syngenta). These populations were tested for 

- 2-
susceptibility to mCry3A and Cry3Bbl using an on-plant assay design (Gassmann et al. 2011) 

and were found to be resistant to both mCry3A and Cry3Bbl. Gassmann et al. suggested that 

cross resistance may be due to structural similarities between mCry3A and Cry3Bbl- both 

toxins are in the three domain (Cry3) family. 

In light of the work by Gassmann and others, the BPPD IRM Team believes that in areas with 

demonstrated Cry3Bbl resistance (e.g., Iowa and Illinois), corn hybrids with mCry3A will 

similarly be at risk for CRW resistance. In addition to single toxin varieties with either Cry3Bbl 

or mCry3A, pyramids with one of these toxins and a 5% refuge (seed blend or structured) 

requirement, may also be at high risk. 

5) Syngenta reported that the company is "exploring options" with regards to developing an onplant assay for resistance detection. However, no further information was provided documenting 

progress towards this goal. Based on results reported by Gassmann et al. (2012, 2014), the 

BPPD IRM Team presumes that MIR604 is compatible with on-plant assay methodology. Given 

the BPPD IRM Team's concerns with the limitations of diet bioassays as resistance detection 

tool, it is recommended that Syngenta initiate on-plant testing (with Gassmann's assay or the 

similar Sublethal Seedling Assay) as soon as possible. It may be useful to continue diet bioassay 

testing on the same populations, so that comparisons can be drawn between the assay 

approaches. 

Background 

Event MIR604 (EPA Registration No. 67979-5), expressing the mCry3A toxin, was approved for 

registration in 2006 and subsequently extended in 2010. Since initial registration, the toxin has 

been registered in several other Bt corn plant-incorporated protectants (PIPs), including stacked 

(e.g., EPA Reg. No. 67979-8: Btll x MIR604) and pyramided varieties (e.g., EPA Reg. No. 

67979-17: Bt11 x 59122 x MIR604 x TC1507), as well as seed blends (e.g., EPA Reg. No. 

67979-20: Btll X 59122-7 X MIR604 X TC1507). 

The mCry3A toxin is active against the com rootworm (CRW) complex, including western com 

rootworm (WCRW, Diabrotica virgifera virgifera) and northern corn rootworm (NCRW, 

Diabrotica barberi). As part of the terms of registration, Syngenta, the registrant of MIR604 Bt 

corn products, must monitor CRW populations for potential resistance to mCry3A. 

The monitoring efforts have included two components: 1) annual sampling and bioassays of 

CRW populations, and 2) investigations of unexpected CRW· damage in mCry3A fields. Past 

monitoring efforts and data (through the 2011 growing season) with mCry3A have been 

reviewed and summarized by EPA (see BPPD 2010a, b; BPPD 2013a, b). As described in those 

reviews, CR W susceptibility has been assessed using diet-based bioassays but has been 

complicated by high variability in response to mCry3A and an apparent lack of a functional 

diagnostic assay to detect resistant populations. The BPPD IRM Team recommended that 

Syngenta consider the Sublethal Seedling Assay developed by Nowatzki et al. (2008) as an 

alternative to testing with artificial diets. 

In 2010, the original MIR604 Bt com registrations (EPA Reg. Nos. 67979-5 and 67979-8) were 

extended until 2015. As part of this extension, several terms were included to improve the CR W 

- 3-
resistance monitoring program. Similar provisions have also been required for other products 

containing mCry3A that have been more recently registered (e. g., 67979-1 7). These terms 

include the following (paraphrased from BPPD 2010c): 

• Development of functional diagnostic assay to detect potentially resistance CRW 

individuals. As part of the efforts, the registrants were required to investigate the 

feasibility of using the Sublethal Seedling Assay (Nowatzki et al. 2008). 

• Development and implementation of a monitoring plan for northern com rootworm 

(NCRW). This plan must include insect sampling and testing ofNCRW susceptibility to 

mCry3A. 

• Revised CRW damage guidelines for mCry3A com (for reports of unexpected pest 

damage in the field) incorporating BPPD's comments from the June 30,2010 review (see 

BPPD 2010a). 

• Investigation of grower, extension specialist or consultant reports of unexpected damage 

or control failures for corn rootworm. 

Summary of Corn Rootworm Resistance Monitoring Submissions for mCry3A 

Syngenta made two submissions to address resistance CRW monitoring requirements in 2012, 

which are detailed below. Each of these monitoring submissions (annual testing with diet 

bioassays and unexpected pest damage reports) have been reviewed separately in this 

memorandum. These sections and the BPPD IRM Team's review of the overall CRW 

monitoring plan are described below. 

mCry3A Bioassay Results: 

MRID# 492065-01: Monitoring Susceptibility of Diabrotica virgifera virgifera to 

mCry3A: 2012 Insect Collections 

Unexpected Damage Reports: 

MRID# 493572-01: Annual Monitoring: 2011 Cropping Year Unexpected Damage 

Reporting; Diabrotica virgifera virgifera and Event MIR604 in 

corn 

MRID# 493572-02: Annual Monitoring: 2012 Cropping Year Unexpected Damage 

Reporting; Diabrotica virgifera virgifera aJ?.d Event MIR604 in 

corn 

A. mCry3A Diet Bioassays 

In accordance with the monitoring plan for mCry3A (MRID# 473401-01; see description in 

BPPD 2010a, b), Syngenta annually samples and screens CRW populations for changes in 

susceptibility to the toxin. Sampling has been coordinated by ABSTC for all Bt corn registrants 

from three defined regions covering different WCRW variants. The regions also represent areas 

of high Bt com adoption and CRW pressure. Region 1 (rotation resistant variety) includes 

-4-
Indiana and eastern Illinois; Region 2 (wild type) consists of western Illinois, Iowa and Missouri; 

Region 3 (organophosphate resistant variant) encompasses Nebraska and Kansas. A minimum 

of2,000 adults are to be collected to establish each population for susceptibility testing. 

Susceptibility to the toxin has been assessed with artificial diet bioassays. In previous testing 

(2006 - 2008), Syngenta used mortality (LCso and LC9o) to assess susceptibility; starting in 2009 

the company also included sublethal measures (i.e., ECso growth inhibition) in addition to 

determining LCsos. 

2012 Sampling and Assays (MRJD# 492065-01) 

In 2012, 13 populations were collected, nine of which produced sufficient offspring for the 

bioassays. CRW were sampled from Iowa (4 populations), Minnesota (1 population), and 

Nebraska (4 populations). Specific county locations of the collections are documented in Table 

1 below. A mCry3A-susceptible control population (from Slater, lA; provided by Syngenta) was 

also included in the assays. Eggs from the sampled adults (collected July- August 2012) were 

kept in diapause until testing (initiated April2013). Once removed from diapause, eggs were 

sterilized and incubated until neonate hatching. 

Testing was conducted by Custom Bio-Products M~well, IA) using artificial diet supplied by 

Syngenta (contained in 96 well bioassay trays). The mCry3A toxin was also provided by 

Syngenta and consisted of a lyophilized powder preparation (concentration 71.4 J...lg protein/mg; 

lot# L 1197054). Serial dilutions of mCry3A were prepared and overlaid on the diet trays to 

achieve the following toxin levels: 0, 0.3, 0.9, 2.7, 8.1, and 24.3 J...lg mCry3A/cm2 diet surface. 

Both the toxin lot source and serial dilution concentrations were used in the 2011 mCry3A 

bioassays. Neonate WCRW were exposed to the serial dilutions (one larvae per well) and 

allowed to'feed for six days after which mortality and (survivor) weight were recorded. Four to 

six valid replicates per population were run with replicates considered invalid if the control 

mortality exceeded 25%. Mortality data were used to determine a LCso value for each 

population by probit analysis (higher LCso determinations were presumably made by 

extrapolation). Larval weight data were grouped and converted to percent growth inhibition 

(relative to the control) to determine ECso values. 

Results from the bioassays are detailed in Table 1 below. Overall ECso susceptibility to mCry3A 

was 0.30- 7.05 J...lg/cm2 for the field-collected populations. One population, from Floyd County, 

lA, was a statistical outlier with the highest ECso (7.05 J...lg/cm2

) ; the ECso range for the other 

tested populatio~s was 0.03- 1.45 J...lg/cm2. LCso values ranged from 1.19 to 8.16 J...lg/cm2

, again 

with the Floyd County population as an outlier with a LCso of 65.20 J...lg/cm2

• The control colony 

was tested at two different times and had ECsos of0.68 (May) and 0.74 J...lg/cm2 (June); the LCso 

for the May test run was 1.12 J...lg/cm2 (the June test data did not converge to produce an LCso). 

Syngenta noted that the results from Floyd County could be "indicative of a shift in susceptibility 

to the mCry3A protein." The company indicated that the location and surrounding area would be 

targeted for more intensive sampling and testing. 

- 5 -
Table 1. Western com rootworm susceptibility to mCry3A using overlay diet bioassays: 2012 

seas ( bl d d fr T bl 1 · S ' 2012 MRID# 492065 01) . on ta e repro uce om a e m ;yngenta s report- - Population Replicates MeanECso 

(95% Cl) 

{J.lg/cm2) 

Hamilton County, IA 6 0.37 

(0.19 - 0.55) 

Floyd County, lA 5 7.05 

(3.49- 20.68) 

Mower County, MN 5 0.62 

(0.42- 0.83) 

Cerro Gordo County, IA 6 1.45 

(0.95- 2.14) 

Stanton County, NE 5 1.26 

(0. 73 - 1.98) 

York County, NE 6 0.30 

(0.25 - 0.35) 

Howard County, NE 4 0.36 

(0.19- 0.53) 

Platte County, NE 6 0.73 

(0.43 - 1.1 0) 

Clay County, IA 6 1.06 

(0.56- 1.81) 

Range 0.30-7.05 

Laboratory Control (May)' 6 0.68 

(0.33 - 1.23) 

Laboratory Control (June}' 7 0.74 

(0.34 - 1.29) 1 Laboratory colony (provtded by Syngenta) was tested twtce (May and June). 2 LCso could not be calculated because data failed to converge. 

MeanLCso 

(95% CI) 

(J.lg/cm2) 

1.19 

(0.36- 2.47) 

65.20 

(22.35 - 3998.33) 

2.49 

(1.0 1 - 5.46) 

4.10 

(2.29 - 6.95) 

8.16 

(3.95- 20.72) 

1.40 

(0.69 - 2.40) 

2.35 

(1.55- 3.52) 

5.81 

(2.99- 12.65) 

4.63 

(2.81 - 7.44) 

1.19-65.20 

-- 2 

1.12 

(0.24- 2.66) 

Table 2. Cumulative Results ofmCry3A Susceptibility Testing for WCRW (2006- 2012; Table 

created from data submitted by Syngenta). 

Year LCS{) range (ng/cm2) LC9o range (ng/cm2

) MRID Citation 

2006 357.9- 1923.6 3788.7- 28,766.6 473401-01 

2007 209.8 - 1172.3 2470.5 - 52,515.0 480470-02 

2008 191.9-4822.61 1430.0- 37,820,880.01 480470-02 

Year ECso range (J..lg/cm2

) LCso range (J..lg/cm2

) MRID Citation 

2009 0.24- 1.14 1.30 -15.31 noMRID# 

2010 0.19 - 1.53 1.49 - 13.96 485913-01 

2011 0.24-3.12 0.53-28.83 489634-02 

2012 0.30 -7.05 1.19-65.20 492065-01 1 .. . .. One populauon m 2008 ex.htbtted htgh tolerance to mCry3A. The other sampled populattons had suscepttb!ltty 

ranges of 191.9- 1518.4 (LCso) and 1430.0 - 225,779.9 (LC90) nglcm2. 

- 6-
BPPD IRMTeam Review- 2012 Diet Bioassays 

Growth inhibition (ECso) testing showed that the populations collected in 2012 were generally 

sensitive to mCry3A, with the exception of one population(Floyd County, lA). Field 

populations responded similarly to the laboratory control colony (Table 1) and CRW collected 

from previous seasons (Table 2). The results from mortality-based testing (LCso) were also 

consistent·in 2012, though all tested field populations had higher values than the laboratory 

control. There is no indication, however, that these colonies (except Floyd County) are resistant 

to the toxin, as high variability in response has been often observed in Bt diet bioassays for CR W 

(see BPPD 2011, 2012, 2013). 

The Floyd County, IA population exhibited approximately 6 to 10 fold higher growth inhibition 

(ECso = 7.05 11g/cm2

) and lethality (LCso = 65.20 11g/cm2

) than the other tested populations (ECso 

range= 0.03- 1.45 IJ.g/cm2

; LCso range= 1.19 - 8.16 jlg/cm2

). The BPPD IRM Team agrees 

with Syngenta's characterization of the population as indicative of a potential shift in 

susceptibility to the mCry3A toxin. Syngenta did not designate the population as "resistant," 

though the resistance ratio (relative to the control population) was 58.2 (Table 1 in MRID# 

492065-01). The BPPD IRM Team also agrees with Syngenta's plan to intensively sample and 

test CR W from the area in subsequent years; presumably this was performed during the 2013 

growing season (data from 2013 had not yet been submitted at the time of this review). It is not 

clear whether any additional investigation was planned, or if the population was maintained for 

further study. 

The Floyd County population highlights the BPPD IRM Team's ongoing concern with the use of 

diet bioassays to make resistance determinations. The population is clearly significantly less 

susceptible to mCry3A than any other field population tested since 2008. Yet, it is not clear 

what level of response would be needed to deem the p0pulation as mCry3A-resistant. The terms 

of registration for mCry3A products (BPPD 2010) indicate that resistance would be confmned if 

"the LCso of the test population exceeds the upper limit of the 95% confidence interval for_the 

LCso of a standard unselected population and/or survival in the diagnostic assay is significantly 

greater than that of a standard unselected population, as established by the ongoing ba5eline 

monitoring program. •• The LCso criterion appears to have been met by the Floyd County 

population, though. the registration definition of resistance implies that the suspect CR W are 

collected from damaged fields. To the BPPD IRM Team's knowledge, the Floyd population did 

not originate from a damaged mCry3A field. 

Much of the BPPD IRM Team's concern with diet bioassays is that ECsoiLCsos cannot reliably 

be used to identify resistant populations. Nor is there a functional "diagnostic" diet bioassay 

capable of distinguishing resistant and susceptible individuals or populations (BPPD 2013b). 

Syngenta's report indicated that they are "exploring options" for on-plant assays as a diagnostic 

tool, but no further information was provided on their progress towards developing a functional 

assay. Based on results reported by Gassmann et al. (2012, 2014), the BPPD IRM Team 

presumes that MIR604 is compatible with on-plant assay methodology. The BPPD IRM Team 

recommends that Syngenta initiate on-plant testing (with Gassmann's assay or the similar 

Sublethal Seedling Assay) as soon as possible. It is also recommended that as part of this effort, 

- 7-
Syngenta test the Floyd County population using an on-plant assays to assess potential 

resistance. 

B. Field Damage Reports 

Syngenta provided reports documenting investigative activities of MIR604 fields with 

unexpected CRW damage. Separate reports were submitted for the 2011 (MRID# 493572-01 ) 

and 2012 (MRID# 493572-02) growing seasons. In addition, an independent researcher 

conducted investigations of CRW populations sampled from damaged mCry3A fields in 2011 

(Gassmann et al. 20 14). 

2011 Damage Reports 

Syngenta reported that the company investigated 12 cases of field damage in which damage to 

MIR604 plants exceeded the current trigger {mean Node Injury Score > 1.0). The damaged 

fields were located in Iowa (8 locations), Nebraska (2 locations), and Illinois (2 locations). CRW 

beetles were collected from each site, though the colonies from four of the locations failed to 

produce enough viable eggs for testing. Populations that could be tested (listed in Table 3 

below) were assessed using diet bioassays and the procedures described in Section A of this 

review. The control colony (Slater, IA; provided by Syngenta) was the same as used in the 

annual monitoring. 

Results from the assays are detailed in Table 3 below. The ECso range for the tested populations 

was 0.72-3.76 Jlg/cm2 while the LCso range was 2.12-13.11 Jlg/cm2 . By comparison, the 

control colony had an ECso of 0.59 Jlg/cm2 and an LCso of2.06 Jlg/cm2

, resulting in (LCso) 

resistance ratios of 1.02 to 6.36 in the field populations. Syngenta noted that the susceptibility 

results from these populations were comparable to the field populations tested as part of the 2011 

annual monitoring program (see Table 2 above or BPPD 2013a). 

In light of the assays results and corresponding resistance ratios, Syngenta concluded that the 

field damage was not likely due to decreased susceptibility to mCry3A. Rather, factors such as 

high CRW density, late planting, or other environmental stressors were more likely causes of the 

field effects. The company indicated that no further investigation on the populations would 

occur but that best management practices were recommended for those locations. 

2012 Damage Reports 

During the 2012 season, Syngenta investigated three damaged fields that resulted in CRW beetle 

collection. All three of the damage sites were located in Sioux County, IA. One population did 

not produce a sufficient number of viable eggs and was not tested. The other two populations 

were assessed using diet bioassays with the procedures described in Section A of this review. 

The control colony (provided by Syngenta) was the same as used in the 2012 annual monitoring; 

the "June" test run was used as the reference point since the "May" test run failed to produce an 

LCso estimate. · 

- 8-
Results from the two tested populations (Hull and Rock Valley) produced ECso values of 1.28 

and 1.04 J..lg/crri2 (Table 3). LCso values for these populations were 6.08 and 42.44 J..lg/cm2. The 

Rock Valley pop:ulation LCso result corresponded to a resistance ratio of37.89. Syngenta stated 

the Hull population was consistent with other monitored CRW populations in 2012 (Table 1), 

while the Rock Valley population could have "a degree of tolerance" to the toxin. The company 

indicated that best management practices were recommended for both locations. It is unclear if 

any additional investigation or sampling was planned for the Rock Valley site/population. 

Table 3. Susceptibility ofCRW collected from MIR604 fields with unexpected damage in 2011 

and 2012 (table reproduced from Syngenta's reports- MRID#s 493572-01 and -02) 

Population Replicates MeanECso MeanLCso 

(95% Cl) (95% Cl) 

(Jlg/cm2) (llg/cm2

) 

20 II Unexpected Damage Sites 

Templeton, lA 4 1.03 7.96 

(0.42 - 1.93) (3.07 - 21.64) 

Swaledale, IA 5 1.51 3.69 

(0.42- 4.93) (2.22 - 5.78) 

Thornton, lA 6 3.76 13.11 

(2.16-6.80) (8.20 - 22.92) 

Lindsay, NE 5 0.88 2.12 

(0.23- 2.99) (1.27 - 3.30) 

Stanton, NE 6 1.83 5.87 

( 1.41- 2.37) (3.20 - 9.12) 

Maryville, lA 6 0.72 3.69 

(0.31 - 1.28) (1.87 - 6.61) 

Prophetstown, IL (Comielus 6 1.18 I 

Field) (0.59 - 2.1 0) --

Prophetstown, IL (Wyffels 6 1.09 4.63 

Field) (0.71- 1.57) (2.40 - 8.50) 

Laboratory Control 6 0.59 2.06 

(0.48- 0.71) (1.50 - 2.74) 

20 I2 Unexpected Damage Sites 

Sioux, lA (Hull) 6 1.28 6.08 

(1.08- 1.5ll (3.29- 10.12) 

Sioux, IA (Rock Valley) 6 1.04 42.44 

(0.49- 1.92) (15.88 - 301.63) 

Laboratory Control 7 0.74 1.12 

(0.34- 1.291 (0.24 - 2.66) 

1 LC50 could not be calculated because data fa1led to converge. 

Gassmann eta/. (2014) 

A separate investigation ofCRW populations sampled from damagedmCry3A fields was 

undertaken by Dr. Aaron Gassmann oflowa State University. Three CRW populations were 

collected from severely damaged fields (NIS > 2.0) in northern Iowa during the 2011 season (six 

other populations were collected from damaged Cry3Bb1 fields). These populations were tested 

for susceptibility to mCry3A and Cry3Bb1 using an on-plant assay design (Gassmann et al. 

2011). 

- 9-
The tested populations were found to be resistant to both mCry3A and Cry3Bbl, as evidenced by 

equivalent survival on Bt and non-Bt com. Gassmann et al. suggested that the cross resistance 

may be due to structural similarities between mCry3A and Cry3Bb1- both toxins are in the three 

domain (Cry3) family. 

It is unclear whether any of the damage sites assessed by Gassmann were also investigated by 

Syngenta as part of their 2011 monitoring efforts. 

BPPD IRM Team Review - Unexpected Damage Investigations 

The BPPD IRM Team agrees with Syngenta that the eight populations collected from damaged 

fields in 2011 appeared to still be susceptible to mCry3A as measured by ECsos in diet bioassays. 

High pest pressure or environmental factors are reasonable explanations for the damage. 

However, separate investigations by Gassmann et al. (2014) during 2011 revealed mCry3Aresistant populations from damaged fields. Therefore, the BPPD IRM Team recommends that 

future testing of CR W from damage sites include on-plant assays (in addition to diet bioassays) 

to buttress resistance assessments. 

One population collected in 2012 (Sioux, IA- Rock City) could also have been resistant to 

mCry3A. This population had an ECso comparable to other tested populations but the LCso value 

was almost 38 fold higher than the susceptible control colony. The use of an on-plant assay 

would also have been beneficial here, particularly given the somewhat ambiguous results of the 

diet bioassays. Several populations in 2011 also had significantly higher LCso values than the 

control, though none of these populations exceeded the 1 0 fold resistance ratio (LCso field 

population/LCso control) typically used to define resistance (Tabashnik 1994). The BPPD IRM 

Team also notes that LCso determinations for CRW have been highly variable (BPPD 2013a), 

often because the results have to be extrapolated from lower concentrations, and may not be a 

reliable measure of potential resistance. 

The work by Gassmann et al. (2014) demonstrated field resistance to both mCry3A and Cry3Bb1 

among nine CR W populations collected from damaged Bt fields. Other researchers have also 

previously reported cross resistance between Cry3Bb1 and mCry3A (Meinke et al. 2012, Zukoff 

and Hibbard 2012; described in BPPD 2013a). Gassmann et al. (2014) suggested that 

phylogenetic similarities between mCry3A and Cry3Bb1 (both belong to the three domain Cry 

family) may be responsible for the observed patterns of cross resistance. On the other hand, 

Cry34/35, which has not shown cross resistance, is a binary toxin that is structurally dissimilar to 

mCry3A and Cry3Bbl. 

In light of the work by Gassmann and others, the BPPD IRM Team believes that in areas with 

demonstrated Cry3Bbl resistance (e.g., Iowa and Illinois), com hybrids with mCry3A will 

similarly be at risk for CRW resistance. In addition to single toxin varieties with either Cry3Bb1 

or mCry3A, pyramids with one of these toxins and a 5% refuge (seed blend or structured) 

requirement, may also be at high risk. There are currently 13 pyramids registered for 

commercial use in which either mCry3A or Cry3Bbl is combined with Cry34/35, though there 

are no registered pyramids containing both mCry3A and Cry3Bbl (BPPD internal analysis). 

- 10-
References 

BPPD, 2010a. Review ofSyngenta's Com Rootworm Monitoring Plan and 2006 Baseline 

Susceptibility Data for MIR604 Com. J. Martinez memorandum toM. Mendelsohn, June 30, 

2010. 

BPPD, 2010b. Biopesticide Registration Action Document: Modified Cry3A Protein and the 

Genetic Material Necessary for their Production (Via Elements of pZM26) in Event MIR604 

Com SYN-IR604-8. Revised September, 2010. Available at 

http://v..'\vw.epa.gov/pesticides/biopesticides/pips/mcry3a-brad.pdf. 

BPPD, 2010c. Terms ~d Conditions for Bt Com Registrations, September 30,2010. Available 

at: http:/ /v.ww .epa. go\ /pesticides/biopesticides/pips/bt -com-terms-condi ti on~df. 

BPPD, 2011. Updated BPPD IRM Review of Reports ofUnexpected Cry3Bb1 Damage, 

Monsanto's 2009 Com Rootworm Monitoring Report, and Revised Com Rootworm Resistance 

Monitoring Plan for MON 88017, MON 88017 x MON 810, MON 863, MON 863 x MON810, 

MON 89034 x TC1507 x MON 88017 x DAS-59122-7, and MON 89034 x MON 88017. J. 

Martinez memorandum to M. Mendelsohn, November 22, 201 1. 

BPPD, 2012. BPPD IRM Team review of Monsanto's 2010 com rootworm monitoring data, 

unexpected damage reports for Cry3Bbl expressing Bt Com and academic reports ofCry3Bbl 

field failures as well as com rootworm resistance. J. Martinez and A. Reynolds memorandum to 

A. Sibold, October 11 , 2012. 

BPPD, 2013a. Review of Com Rootworm Resistance Monitoring Submissions and Data for Bt 

com products containing mCry3A. A. Reynolds memorandum to A. Sibold, May 15, 2013. 

BPPD, 2013b. White Paper on Com Rootworm Resistance Monitoring for Bt Plant-Incorporated 

Protectants. September 27, 2013. Available at: 

http :1 /wv..rw.regulations.go\/#! documentDetai l:D=EP A-HQ-OPP-20 13-0490-0008. 

Gassmann, A.J., J.L. Petzold-Maxwell, R.S. Keweshan, and M.W. Dunbar, 2011. Field-evolved 

resistance to Bt maize by western com rootworm. P LoS ONE. 6(7): 1-7. 

Gassmann, A.J., J.L. Petzold-Maxwell, E.H. Clifton, M.W. Dunbar, A.M. Hoffmann, D.A. 

Ingber, and R.S. Keweshan, 2014. Field-evolved resistance by western com rootworm to 

multiple Bacillus thuringiensis toxins in transgenic maize. Proc. Natl. Acad. Sci. USA. 111 (14): 

5141-5146. (doi: 10.1073/pnas.l317179111) · 

Meinke, L., T. Hunt, G. Kruger, R. Wright, D. Wangila, and K. Miwa, 2012. Evaluation of 

western com rootworm susceptibility to rootworm Bt traits in Nebraska. Presentation at the 

Entomological Society of America Annual Meeting. November 13, 2012. 

- 11 -
Nowatzki, T.M., S.A. Lefko, R.R. Binning, S.D. Thompson, T.A. Spencer, and B.D. Siegfried, 

2008. Validation of a novel resistance monitoring technique for com rootworm (Coleoptera: 

Chrysomelida) and event DAS-59122-7 maize. J Appl. Entomol. 132: 177-188. 

Tabashnik, B.E., 1994. Evolution of resistance to Bacillus thuringiensis. Annu. Rev. Entomol., 

39:47-79. 

Zukoff, S.N. and B.E. Hibbard, 2012. Evaluation of the potential development of cross 

resistance between eight transgenic com types in western corn rootworm (Diabrotica virgifera 

virgifera): Presentation at the Entomological Society of America Annual Meeting. November 

13, 2012. 

- 12-