UNITED STATES ENVIRONMENTAL PROTECTIONAGENCY WASHINGTON, D.C. 

### 20460

OFFICE 01- CHF\1lCAl. SAl ETY Al"D POLL! TION PRE\TNT!ON



### MEMORANDUM SUBJECT:



### TO:

FROM:



### PEER REVIEW: Review ofComRootworm Resistance Monitoring Submissions and Datafor Btcomproducts 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. DPBarcodes: 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 Martinez, Ph.D., Ecologist/Entomologist

## ~ Microbial Pesticides Branch Biopesticides and Pollution Prevention Division (751
Action Requested The BPPD IRM Team1

### has been asked to review comrootworm resistance monitoring da ta and informationregarding the mCry3A Btcomtoxin. Submissions were made by Syngenta Seeds, Inc
. (hereafter Syngenta) to. address terms ofregistration for Btcomproducts that express mCry3A in event MIR604, including the Agrisure RWproduct 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.

1The BPPD lRM Team consists ofAlan Reynolds and Jeannette MartinezConclusions and Recommendations 1) Diet bioassays (i.e., growth inhibition, ECso) gene rall y s howed 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 (LCs o ) were also consistent among the 2012 populations, though more variable than the EC so assays. 2) One population collected in 2012 (Floyd County, IA) had approximately 6 to 10 fold higher growth inhibition (ECs o = 7.05 J.!g/cm 2 ) and lethality (LCso = 65.20 j.!g / cm 2 ) than the other tested populations (ECso r ange = 0.03 - 1.45 J.!g/cm 2 ; LCso range = 1.19 - 8.16 J.!g/cm 2 ). 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 (re l ative 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 no t clear from the report whether any additional investigation was planned , or if the population was maintained for further study . The F l oyd 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 I RM 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 viab l e 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 EC so values comparable to other tested populations, though one (Rock V alley, IA) had a LC so value that was almost 38 fold higher than the susceptible control co l ony. Syngenta characterized these results as being mixed, while noting that the Rock Valley population exhibited "a degree of tolerance" to the toxin. The BPPD I RM Team i s 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 mCry 3 A (Gassmann et al. 2014 ) . Three CRW populations were collected from

severely damaged mCry3A fields in northern Iowa during the 20 1 1 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 resistantto bothmCry3A and Cry3Bbl. Gassmann et al. suggested that crossresistance may be due to structural similarities between mCry3A and Cry3Bbl- both toxins are inthe three domain (Cry3) family. Inlight ofthe workbyGassmann and others, the BPPD IRM Team believes that in areas with demonstrated Cry3Bbl resistance (e.g., Iowa and Illinois), cornhybrids with mCry3A will similarlybe atrisk for CRWresistance. In additionto single toxin varieties with either Cry3Bbl ormCry3A, pyramids with one ofthese toxins and a 5% refuge (seedblend or structured) requirement, may also be athighrisk. 5) Syngentareported that the company is "exploring options" withregards to developing an onplant assay for resistance detection. However, no further information wasprovided 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 limita tions ofdiet bioassays as resistance detection tool, it is recommended that Syngentainitiate on-plant testing (with Gassmann's assay orthe similar Sublethal Seedling Assay) as soon as possible. It maybe useful to continue diet bioassay testing onthe same populations, so that comparisons canbe drawn betweenthe assay approaches. Background Event MIR604 (EPA RegistrationNo. 67979-5), expressing the mCry3Atoxin, was approved for registration in 2006 and subsequently extendedin 2010
. Since initial registration, the toxinhas beenregistered in several otherBtcornplant-incorporatedprotectants (PIPs), including stacked (e.g., EPA Reg. No. 67979-8: Btll x MIR604) andpyramided varieties (e.g., EPA Reg. No. 67979-17: Bt11 x 59122 x MIR604 x TC1507), as well as seed blends (e.g., EPAReg. No. 67979

-20: Btll X 59122

-7 X MIR604 X TC1507)
. ThemCry3A 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 ofthe terms ofregistra tion, Syngenta, theregistrant ofMIR604 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 CRWpopulations, and 2) investigations ofunexpected CRW·damage inmCry3A fields. Past monitoring efforts and data (throughthe 2011 growing season) with mCry3A have been reviewed and summarizedby EPA (see BPPD 2010a, b; BPPD 2013a, b). As described inthose reviews, CR W susceptibility has been assessed using diet-based bioassays buthas been complicatedby high variability inresponse to mCry3A and an apparent lack ofa functional diagnostic assay to detectresistant populations. The BPPD IRM Team recommended tha t Syngenta consider the Sublethal Seedling Assay developed byNowatzki et al. (2008) as an alternative to testing with artificial diets. In2010, the original MIR604 Btcomregistrations (EPA Reg. Nos. 67979-5 and 67979-8) were extended until 2015. Aspart ofthis 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 f or 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 une x pected pest damage reports) have been reviewed separately in this memo r andum. 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 acco r dance 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 suscept i bility 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 repres ent areas of high Bt com adoption and CRW pressure. Region 1 (rotation resistant variety) includes -4-Indianaand eastern Illinois; Region 2 (wild type) consists ofwestern Illinois, Iowa andMissouri; Region 3 (organophosphate resistantvariant) encompasses Nebraska and Kansas. A minimum of2,000 adults are to be collected to establish eachpopulationfor susceptibilitytesting. Susceptibility to the toxinhas 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 LCso s. 2012 SamplingandAssay s ( MRJD# 492065-01 ) In 2012, 13 populations were collected, nine ofwhichproduced sufficient offspring for the bioassays. CRW were sampled from Iowa (4 populations), Minnesota (1 population), and Nebraska (4 populations). Specific county locations ofthe collections are documented inTable 1 below. A mCry3A-susceptible control population (from Slater, lA; provided by Syngenta) was also included inthe assays. Eggs from the sampled adults (collected July- August 2012) were kept indiapause 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 suppliedby Syngenta (contained in 96well bioassay trays). The mCry3Atoxin was also providedby Syngenta and consisted ofa lyophilized powderpreparation (concentration 71.4 J...lg protein/mg; lot# L1197054). Serial dilutions ofmCry3A were prepared and overlaid onthe 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. Boththe toxin lot source and serial dilution concentrations were used in the 2011 mCry3A bioassays. Neonate WCRW were exposed to the serial dilutions (one larvaeper well) and allowed to'feed for six days after which mortality and (survivor) weight were recorded. Fourto six validreplicates perpopulation wererun withreplicates considered invalid ifthe control mortality exceeded 25%. Mortality data were used to determine a LCso value for each populationbyprobit analysis (higher LCso determinations werepresumablymade by extrapolation). Larval weight datawere grouped and converted to percent growth inhibition (relative to the control) to determine ECso values. Results from the bioassays are detailed inTable 1 below. Overall ECsosusceptibility 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 thehighest ECso (7.05 J...lg/cm 2) ; the ECso range forthe other tested

**populatio~s** was 0.03- 1.45 J...lg/cm2

. LCso values ranged from 1.19 to 8.16 J...lg/cm 2
, again withthe Floyd Countypopulation as an outlier with a LCso of65.20 J...lg/cm2• The control colony was tested attwo different times and hadECsos of0.68 (May) and 0.74 J...lg/cm2 (June); the LCso for the Maytest run was 1.12 J...lg/cm2 (theJunetest data did not converge to produce anLCso). Syngenta noted that theresults from FloydCounty couldbe "indicative ofa shift in susceptibility to the mCry3A protein." The company indicated that the location and surrounding area wouldbe targeted for more intensive sampling and testing.

- 5 -Table 1. Western co m rootworm susceptibility to mCry3A using overlay diet bioassays : 2012 s eas ( 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, I A 6 0.3 7 (0. 19 - 0.55) Floyd Cou n ty, lA 5 7.05 (3 .49- 20.68) Mower County, MN 5 0.62 (0.42- 0.83) Cerro Gordo C ounty , 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, N E 4 0.36 (0.19- 0.53) Pla tte County, NE 6 0.73 (0.43

- 1.1 0) Cla y County, IA 6 1.06 (0.56- 1.81) Ran ge 0.30-7.05 Lab o rat ory Control (May)' 6 0.68 (0.33

- 1.23 ) Labo rat ory Control (J une}' 7 0.74 (0.34

- 1.29) 1 Labora t ory colo n y ( p rovt d ed b y Syngenta) was tested twtce (May and June) . 2 LCso could not be calculate d because data fail ed t o conve r ge.

MeanLCso (95% CI) (J.lg/cm 2 ) 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 (0.6 9 - 2.40 ) 2.35 (1.5 5- 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 Su sc eptibility Testing for WCRW (2006- 2012; Table created from data submitted by Syng enta) . Year LCS{) range (n g/cm 2 ) LC9o range (ng/ cm 2 ) MRID Citation 2006 357.9- 1923 .6 3788.7- 28,766.6 473401-01

200 7 209.8 - 1172.3 2470.5 - 52,515.0 480470-0 2 2008 1 91.9-4822 .6 1 1430.0- 37,820,880.0 1 4804 70-02 Year ECso rang e (J..l g/cm 2 ) LCso range (J..lg/cm 2 ) 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

201 2 0.30 -7 .05 1.19-65 .20 492065-01 .. . .. 1 One popu lauon m 2008 ex.htbtted htgh tol erance to mCry3A. The other sam p led populattons had suscepttb!ltty ranges of 191.9- 1518.4 (LCso) a nd 1430.0 - 225,779.9 (LC90) nglcm 2 . - 6-

### BPPDIRMTeam Review- 2012 DietBioassays Growth inhibition (ECso) testing showed that the populations collected in 2012 were generally sensitive to mCry3A, with the exception ofone 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·in2012, 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 dietbioassays for CR W (see BPPD 2011, 2012, 2013). The Floyd County, IApopulation exhibited approximately 6 to 10 fold higher growth inhibition (ECso 

## =7.05 11g/cm 2) and lethality (LCso 

## =65.20 11g/cm 2) than the other tested populations (ECso range=0.03- 1.45 IJ.g/cm2; LCso range= 1.19

- 8.16 jlg/cm 2). The BPPD IRM Team agrees with Syngenta's characterization ofthe population as indicative ofa potential shift in susceptibility to the mCry3A toxin. Syngenta did not designate the population as "resistan t," 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'splan to intensively sample and test CR W from the area in subsequentyears; presumably this wasperformed during the 2013 growing season (datafrom 2013 had not yet been submitted atthe time ofthis review). Itis not clear whether any additional investigation was planned, orifthepopulation 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. Thepopulation is clearly significantly less susceptibleto mCry3A than any other field populationtested since 2008. Yet, it is not clear what level ofresponse would be needed to deem the p0pulation as mCry3A-resistant. Theterms ofregistration for mCry3A products (BPPD 2010) indicate that resistance would beconfmnedif "the LCso ofthe test population exceeds the upper limit ofthe 95% confidence interval for

_ the LCso ofa standard unselected population and/or survival in the diagnostic assay is significantly greaterthanthat ofa standard unselected population, as established by the ongoing ba5eline monitoring program.•• The LCso criterion appears to have been metbythe Floyd County population, though
. the registration definition ofresistance implies that the suspect CR W are collected from damaged fields. Tothe BPPD IRM Team's knowledge, the Floyd population did not originate from a damaged mCry3A field. Much ofthe BPPD IRM Team's concern with diet bioassays is that ECsoiLCsos cannotreliably beused to identify resistant populations. Nor is there a functional "diagnostic" dietbioassay capable ofdistinguishing resistant and susceptible individuals orpopulations (BPPD 2013b). Syngenta'sreport indicated that they are "exploring options" for on-plant assays as a diagnostic tool, butno further information was provided ontheir progress towards developing a functional assay. Based onresults reported by Gassmann et al. (2012, 2014), the BPPD IRM Team presumes that MIR604 is compatible with on-plant assay methodology. The BPPD IRMTeam recommends that Syngenta initiate on-plant testing (with Gassmann's assay orthe similar Sublethal Seedling Assay) as soon as possible. It is also recommended that aspart ofthis 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 -0 2) 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 In jury Score > 1 .0). The damaged fields were located in Iowa (8 locations), Nebraska (2 l ocations), 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

annua l monitoring. Results from the assays are detailed in Table 3 below. The ECs o ran ge for the tested populations was 0.72-3.76 Jl g/cm 2 while the LCso range was 2.12-13.11 Jlg/cm 2 . By comparison, the control colony had an ECso of 0.59 Jlg/cm 2 and an LCso of2.06 Jlg/cm 2 , 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 li ght of the assays results and corresponding resistance ra tios, Syngenta concluded that the field damage was not likely due to decreased susceptibi lity 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 investigat ed three damaged fields that resulted in CRW beetle co llec tion. All thr ee 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 20 1 2 annual monitoring ; the "June" test run was used as the reference point since the "May" test run fai l ed to produce an LCso estimate . · - 8-Results from the twotested populations (Hull and RockValley) 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 aresistance 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 "adegree oftolerance" to the toxin. The company indicated that best management practices were recommended for bothlocations. It is unclear if any additional investigation or sampling was planned for the Rock Valley site/population. Table 3. Susceptibility ofCRWcollected 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) 20II UnexpectedDamage 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 UnexpectedDamageSites Sioux, lA (Hull) 6 1.28 6.08 (1.08- 1.5ll (3.29- 10.12) Sioux , IA (RockValley) 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) 1LC50 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 oflowaState University

. Three CRWpopulations were collected from severely damaged fields (NIS 

### > 2.0) in northern Iowa during the 2011 season (six otherpopulations 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 equivalen t 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 o f their 2011 monitoring efforts. BPPD IRM Team Review - Unexpected Damage Investigations The BPPD I RM Team agrees with Syngenta that the e i ght populations collected from damaged fields i n 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 resistan ce 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/LC so 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 reporte d 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 res po nsible 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 ComRootworm 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 Cry3AProtein and the Genetic Material Necessary for their Production (ViaElements ofpZM26) 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 ComRegistrations, September 30,2010. Available at: http:/ /v.ww.epa.go\/pesticides/biopesticides/pips/bt-com-terms-conditi

**on~df**

**.** BPPD, 2011. Updated BPPD IRM Review ofReports ofUnexpected Cry3Bb1 Damage, Monsanto's 2009 ComRootworm Monitoring Report, and Revised ComRootworm Resistance Monitoring Planfor MON 88017, MON 88017 x MON 810, MON 863, MON 863 x MON810, MON 89034 x TC1507x MON 88017 x DAS-59 122-7, and MON 89034 x MON 88017. J. Martinez memorandum to M. Mendelsohn, November 22, 201 1. BPPD, 2012. BPPD IRM Team review ofMonsanto's 2010 comrootworm monitoring data, unexpected damage reports for Cry3Bbl expressing Bt Com and academic reports ofCry3Bbl field failures as well as comrootworm resistance. J. Martinez and A. Reynolds memorandum to A. Sibold, October 11 , 2012. BPPD, 2013a. Review ofComRootworm Resistance Monitoring Submissions andDatafor Bt comproducts containing mCry3A. A. Reynolds memorandum to A. Sibold, May 15, 2013. BPPD, 2013b. White Paper onComRootworm Resistance Monitoring for BtPlant-Incorporated Protectants. September 27, 2013. Available at: http

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. Gassmann, A.J., J.L. Petzold-Maxwell, R.S. Keweshan, and M.W. Dunbar, 20 11. Field-evolved resistance to Btmaizeby western comrootworm. PLoS 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-evolvedresistance bywestern comrootworm 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 comrootworm susceptibility to rootworm Bttraits inNebraska. Pr esentation atthe Entomological Society ofAmerica Annual Meeting. November 13, 2012.

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