2 EXECUTIVE SUMMARY AND CONCLUSIONS

The BPPD IRM team has analyzed annual corn rootworm (CRW) resistance monitoring data submitted by Monsanto for the Cry3Bb1 toxin. These data include populations sampled from the Corn Belt during the 2011 growing season and investigations of unexpected damage in Cry3Bb1 fields in part of the Corn

Belt.

BPPD concluded that the 2011 field populations (collected at random within the Corn Belt) had mean

EC 50 values similar to those exhib ited by the lab reference strain. No comparison could be made to the

historical EC 50 range (2007

- 20010) because Monsanto switched its Cry3Bb1 protein source. BPPD concluded that four samples collected from Cry3Bb1 corn instead of non

- Bt host plants had sig nificantly lower mortality rates and higher average body mass than the reference strain at the highest (diet bioassay) concentration tested. Although Monsanto implemented Best Management Practices during 2012 for these locations (Floyd Co., IA; Hamilton Co ., IA; Buchanon Co., IA; and Stanton Co., NE), BPPD recommends continued monitoring of these sites in subsequent growing seasons.

In 2011, Monsanto investigated CRW populations from fields with unexpected damage using a new on

plant assay methodology (similar to Gassmann et al. 2011). After reviewing those data, BPPD identified one population (Trent, SD) that was likely Cry3Bb1

- resi stant, given that there was no difference in percent mortality, body weight

, and head capsule width between CRW exposed to non

- Bt and Cry3Bb1 plants. T he remaining three populations (Shell Rock, Onslow, and Mankato) had also no significant

difference in su rvival on Bt and isoline corn, while average head width and body weight differed significantly. These results together are inconclusive

, and it cannot be concluded that the other three populations are resistant. BPPD recommends that Monsanto monitor these four locations for further field failures in the coming future.

Monsanto has made significant progress towards developing an on

- plant assay as a diagnostic tool that could be used for future corn rootworm resistance monitoring. BPPD agrees with Monsanto t hat a comparison between the distributions of instar development should provide greater power than a comparison between survival on Bt and non

- Bt seedling mats. BPPD notes, however, that the distributions of the lab and field populations on Bt seedling mat s do not need to be completely non

overlapping before a field population is called resistant. Rather, a statistically significant difference in the two distributions should suffice to make that determination. Furthermore, BPPD pro poses that Monsanto

use a second measure ( i.e., body mass) to confirm resistance. Two independent measures taken together to identify resistant corn rootworm populations should reduce false positives (i.e., concluding a population is resistant when it is not).

In July, 2013, Mons anto’s Cry3Bb1 registrations were amended to include a new resistance detection

strategy. This revised approach incorporates field damage triggers for unexpected crop damage as well as

the use of on

- plant bioassays to determine resistance. These monitorin g procedures were not in place when Monsanto conducted testing of the 2011 CRW populations (tests were performed in 2012); however, Monsanto did conduct on

- plant assays of six populations collected from performance inquiry fields during 2011 (as described above). Though four of the populations appeared to partially meet the criteria for resistance, there was insufficient information to make a conclusive determination.

Preliminary (not yet published) reports from three research teams suggest potential cross resistance between Cry3Bb1 and mCry3A. A number of cases of field

- evolved Cry3Bb1 resistance have been recently documented (e.g., Gassmann et al. 2011) and in some cases, Cry3Bb1

- tole rant populations have3 also shown decreased susceptibility to mCry3A (Ga ssmann et al. 2012, Meinke et al. 2012, Zukoff and Hibbard 2012). The BPPD IRM Team recognizes that additional data will be needed to fully assess potential cross resistance and implications for Insect Resistance Management.

Monsanto provided an update o n the resistance monitoring program for northern corn rootworm (NCRW) which demonstrated that there are significant logistical challenges including difficulties with sampling, rearing, egg production, and testing with bioassays. For this reason, the BPPD I RM team concurs with industry that, at the present time, it is not possible to effectively monitor NCRW for changes in toxin susceptibility. Despite the technical hurdles presented by this pest, BPPD continues to recommend that Bt corn registrants work to develop a functional resistance monitoring program for this insect and that ABSTC continue their efforts to collect NCRW from representative regions of the Corn Belt.

RECOMMENDATION S

Monsanto noted that some methodological changes for diet bioassays were made in addition to switching the protein source and testing of a new highe st concentration. It was unclear to BPPD what those additional changes were. BPPD recommends that Monsanto specifically communicate all modifications of resistance monitoring methodologies

.

When body weight results are obtained with Bt diet bioassay for the purpose of determining whether a corn rootworm population is resistant, BPPD recommends that the comparisons be made between the field population and a resistant reference colony rather than the susceptible control colony. More information can be extracted from the first comparison.

BPPD recommends that the registrant use a second measure for sublethal seedling assays to confirm

resistance ( i.e. , body mass)

.

Monsanto inve stigated the feasibility of a single on

- plant assay (using MON 88017 seedlings) for 2011

pro blem fields and fields in high

- pressure areas. BPPD recommends that Monsanto also consider using MON 862 plants for on

- plant assays because of the greater toxin exp ression

, which c ould allow further

separat ing resistant from susceptible individuals.

The registrant chose to measure decreased Cry3Bb1 susceptibility with “percent (larval) recovery” for on

plant assays. BPPD recommends that Monsanto use percent mortality for on

- plant assays because “percent larval recovery” has not been a reliable meassure for identifying putative resistant from susceptible individuals. Head capsule width and average dry weight were two additional measures that showed greater pro mise in separating susceptible from resistant populations. Some aspects of the methodology were not described in Monsanto’s submission, and it was

, therefore, unclear how similar

Monsanto’s assay was to Dr. Gassmann ’s

. If applicable, BPPD recommends that M onsanto consider using a 17

- day assay period since this duration allowed for insects to reach third instar development Gassmann et al. (2011)

.

BPPD concludes that while Monsanto’s BMPs are a reasonable mitigatory approach to dealing with unexpected damag e in Bt corn fields, most of the BMPs are not a long

- term solution for managing Cry3Bb1resistance and/or putative resistance in corn rootworm (except crop rotation and switching to another Bt trait). Furthermore, if Cry3Bb1 resistance is known to have evol ved in a particular population or region , continued planting of the compromised toxin or pyramid s contain Cry3Bb1 deployed with a 5%4 refuge is not recommended

. To protect the longevity of a single toxin, theoretical modeling supports increasing the seed bl end to a minimum of 10% (BPPD 2010 a ).

I. BACKGROUND

Corn rootworm ( CRW, Diabrotica spp. ) is among the most serious economic insect pests of corn in the United States (Levine & Oloumi

- Sadeghi, 1991). Western corn rootworm ( Diabrotica virgifera virgifera

, WCRW ) is found from Mexico to the U.S. Corn Belt and Canada and is widely distributed; Northern corn rootworm ( D. barberi

, NCRW ) is found in the Midwest of the U.S and has a localized distribution. Together, they are the most prevalent Diabrotica pests in the U

. S. The Mexican corn rootworm ( D. virgifera zea , MCRW ) has a distribution from Central America to the southern U.S. and is a sporadic problem in central Texas and southern Oklahoma.

In February 2003, the Agency approved the first Bacillus thuringien sis ( Bt ) toxin targeting corn

rootworm: Monsanto’s Cry3Bb1 protein ( event MON 863 ( YieldGard Rootworm, EPA Reg. No. 525

- 528)) and the genetic material (Vector ZMIR13L) necessary for its production. Cry3Bb1 was the first Bt PIP product registered to protect crops against corn rootworm (CRW) species.

In December 2005, MON 88017 was approved for registration. The Cry3Bb1 toxin expressed in MON

88017 is equivalent ( 99.8% similar ) to that in MON 863. The Cry3Bb1 protein produced in MON 88017

and MON 863 is a v ariant of the wild

- type Cry3Bb1 protein from Bt subspecies kumamotoensis

. The primary difference between the hybrids is that MON 88017 also expresses a gene for resistance to glyphosate (Roundup) based herbicides.

One “pyramided” Bt corn product has been registered that includes Cry3Bb1. SmartStax is a stacked PIP product consisting of a three

- gene pyramid (Cry1A.105xCry2Ab2xCry1F) targeting lepidoptera and a

two

- gene pyramid (Cry3Bb1xCry34Ab1/35Ab1) aimed at contr olling corn rootworm. It was conditionally re gistered by the Agency in July 2009 with a 5% structured refuge for lepidoptera and corn rootworm. Subsequently, the Agency approved registration of a SmartStax product with a 5% seed blend (“refuge

- in

- the

- bag ”). At present, there are no other pyramided products that contain the Cry3Bb1 toxin.

At the time of this review , there are three other registered Bt toxins active against CRW (mCry3A, Cry34Ab1/35Ab1, and eCry3.1Ab). Similar to Cry3Bb1

, none of these to xins express a high dose against

corn rootworm

. Simulation modeling predict s and empirical studies confirm that resistance evolves

fastest when the domin ance of the resistance allele i s increased by lowering the dose of toxi n. The greater

risk of resistan ce evolution to Bt crops can be expected when toxins are less than high dose ( Roush 1998, Tabashnik et al., 2004). The first a cademic reports of corn rootworm resistance to Cry3Bb1 emerged in 2009, six years after the c ommercialization of the toxin

.

In th e 2012 resistance monitoring review for Cry3Bb1, BPPD concluded that as in previous years, Monsanto’s diet bioassays showed that field

- collected populations (collected from Illinois, Iowa, Minnesota, and Nebraska) had generally higher EC 50 values than th e susceptible laboratory colony although the values (with one exception) fell within the range o f previous years’ collections (BPPD 2012)

. The BPPD IRM team’s analysis of monitoring data from 2007 to 2010 indicate d a general trend of reduced susceptibility to Cry3Bb1. There were concerns, however, that the diet bioassay approach was likely insufficient to reliably detect resistant populations in a timely manner

. BPPD also summarized5 reports of Cry3Bb1 corn rootworm resistance reported by academia in Iowa in 2011

- 2012 and in two

counties in western Illinois during the 2011 growing season

. The BPPD IRM team concur red with the conclusions of the researchers that the tested CRW collections from Iowa and Illinois represent ed Cry3Bb1

- resistant populations ; the se populations did, however, not meet the current regulatory definition of resistance (see discussion in BPPD 2012) . To improve the current Cry3Bb1 monitoring prog ram the

BPPD IRM team recommended the following enhancements be adopted :

• Expansion of the a nnual sampling plan to include the states of Wisconsin, South Dakota, and Colorado. Corn rootworm damage to Cry3Bb1 fields has been reported in parts of these three states.

• The use of high risk factors (causal factors of resistance) to determine annual sa mpling locations.

These considerations should include areas in close proximity to reported field failure/unexpected rootworm damage, fields with a history of corn

- on

- corn planting ( i.e., lack of crop rotation with a

non

- CRW host plant), repeated ( e.g., pr ophylactic) use of Cry3Bb1, and/or high non

- compliance with refuge requirements.

• Standard use of an on

- plant assay (such as those developed by Gassmann et al. 2011 and /or Nowatzki et al. 2008) in lieu of diet bioassays as the primary resistance detection t ool.

In 2010, the terms and conditions of all Bt corn registrations were amended to require that registrants with corn rootworm PIP registrations investigate the feasibility of using a sublethal seedling assay as a diagnostic assay. A progress report tow ards this requirement needed to be submitted within six months

from the date of the amended registration (BPPD 2010b). Monsanto has subsequently su bmitted several

reports ( Letter s dated 8

- 31

- 2012 and 9

- 28

- 2012, MRID s 484368

- 01 and 486367

- 01)

, in which the

company further communicated their progress towards an on

- plant diagnostic assay. Additionally Monsanto submitted their 2011 rootworm resistance monitoring report for Cry3Bb1. Monsanto’s latest

submission s are summarized and discussed in this review.

II. BPPD SUMMARY OF MONSANTO’S SUBLETHAL SEEDLING ASSAY PROGRESS

REPORT S AND UPDATE ON THE CONDITIONS OF REGISTRATION FOR MON

88017

a. Monsanto 3/3/ 2011 submission ( MRID 484368

- 01 )

Monsanto’s report was submitted in response to BPPD’s request that the registrant detail their progress on developing a sublethal seeding assay methodology (BPPD 2010a). In this first submission Monsanto asserted that MON 88017 seedlings were not practical for use in SSA because western corn rootworm larval development (morta lity and development) was less affected than what was observed in the field. The experimental design employed in method development followed other currently published and unpublished pr ocedures (Nowatzki et al. 200 8 and Head & Storer 2008). Seeds of MON 88017 and non

Bt corn were planted in separate p lastic containers and grown under appropriate growth chamber conditions. Larvae (or eggs) from susceptible colonies and one resistant weste rn corn rootworm colony (Hibbard, USDA

- ARS lab) used to infest the root mats in the plastic containers. Developing larvae were

allowed to feed for 5

- 10 days before they were extracted from the root mats with Berlese funnels. Larvae

were collected into vess els containing an ethanol solution, counted, and their head width and instar

determined. Afterwards larvae were freeze dried and their weight s were determined. The initial results using this methodology were, however, inconclusive. Differences in mortality and development6 parameters measured on rootworm larvae from Cry3Bb1 and non

- Bt plants were not great enough to clearly discriminate susceptible an d resistant larvae on Bt treatments. Method development was on

- going at the time of the report

.
In response to BPPD’s sampling concerns (BPPD 2010a), Monsanto reported that ABSTC ’s 2010 adult

collections were expanded to include Minnesota and further area s in Iowa. Additionally, the registrant

also made collection in northeastern Iowa and two locations in South Dakota during the 2010 season

.
As indicated in the terms and conditions of Monsanto’s Cry3Bb1 registration, BPPD requested that

research be conducted to determine if IRM strategies developed for NCR W and WCRW were appropriate for the Mexican rootworm (MCR) (BPPD 2010a). Monsanto reported that all available data continue to

indicate that WCRW and MCR are genetically very similar (e.g., Szalansk i et al., 1999), though WCRW is more abundant and widespread making WCRW a natural surrogate for MCR. Where possible, Monsanto has conducted efficacy testing in areas expected to have MCR to confirm that the efficacy of Monsanto's corn rootworm products ag ainst MCR is comparable to that against WCRW (e.g., Moore, 2004), and therefore that

WCRW is a reasonable surrogate for MCR. The efficacy of these products has been uniformly good in these areas, indicating that efficacy against MCR is indeed comparable to efficacy against western corn rootworm

. However, rootworm pressure is consistently light in these areas making field testing and collecting MCR, problematic. Furthermore, rearing methods have not been worked out for MCR

, as is the case for NCR W and,

there fore

, field efficacy work remains the logical focus for MCR assessments.

b. Monsanto 9/28/2011 submission (MRID 486367

- 01)

Monsanto indicated that MON 88017 seeds did not allow for successful discrimination between Cry3Bb1 resistant and susceptible insects and determined tha t seeds from a different event, such as MON 862

, had much higher toxin expression levels than MON 88017 seeds and, therefore, showed grea ter promise to

better discriminate between susceptible and putative resistant insects. Hence, a ppropriate changes were made to the experimental protocol described in MRID 484368

- 01 to include the different seed source

from MON 862

. Root expression data fro m MON 862 and MON 88017 are shown at different growth stages in Table 1 below

. MON 862 seeds were limited in the next trial, and therefore only 15 Bt and non

Bt seeds were planted per treatment container. Infestation and duration of allowed feeding was ini tiated as

described in the earlier report as well

.

The experimental design of the SSA was modeled after Nowatzki et al. (2008) and Head & Storer (2008)

. SSA results with MON 862 seedlings showed great promise ( see Table 2 ) . The percent larval recovery 10 days after infestation was very low in Bt treatment containers for larvae from susceptible colonies but was 36 to 52

- fold higher in non

- Bt treatment containers . In comparison, resistant larvae (USDA lab selected colony) were reco vered in similar numbers from Bt and non

- Bt seedling mats as expected. In both cases,

t he percent recovery of resistant larvae was similar to the percent recovery of susceptible larvae from non

Bt seedling mats. T he response of resistant and susceptible la rvae on the Bt seedling mats was very different (see Figure 1 )

. L arval recovery was low for the susceptible larvae indicating that larval recovery does not appear to be a suitable metric for this assay system if the discrimination between resistant and susceptible larvae is to be effective.

The observed age structure percentages of surviving resistant and susceptible larvae on MON 862 seedling mats were non

- overlapping (see Table 3 )

. Susceptible larvae (both colonies) did not develop to

third instar

, while 65% of the USDA resistant colony reached third instar by the end of the assay ( see Figure 2 ). Monsanto concluded that with MON 862 the difference in development between resistant an d7 susceptible populations might be large enough to consistently discriminate between susceptible and putative resistant larvae.

Since the MON 862 seed source was limited, another comparable Cry3Bb1 expressing line was used to

test whether it could successfully replace MON 862 . The registrant continue d to improve and optimize the SSA methodology . Monsanto ha d further concerns that the greenhouse selected USDA resistant colony might not reasonably represent the resistance encountered in the field. Hen ce
, the registrant ha d collaborated with Dr. Aaron Gassmann from Iowa State University to obtain additional resistant western

corn rootworm colonies

.

Monsanto also updated BPPD that the rearing and laboratory testing of MCR and NCR remained diffic ult

and problematic. A vailable data continue d to indicate that all three corn rootworm species were genetically and biologically very similar

, and control of MCR on Cry3Bb1 expressing corn was similar t o the control observed for Western corn rootworm. WCRW , howeve r, remain ed the most abundant and widespread species of the corn rootworm complex.

An expert meeting was organized by member companies of ABSTC at the 2012 Entomological Society Meeting in Reno, NV, to discuss rearing and bioassay methods for NCRW and wh ether they could be improved successfully to allow monitoring for resistance in NCR. Monsanto’s submission indicated that a

summary report would be provided to the EPA (summarized in next section)

.

Table 1 Expression Levels of Cry3Bb1 in MON 862 and MON 88017 Root Tissue s

Seed Type Growth Stage Av g

. Expression Level (ppm of Cry3Bb1)

MON 88017 V2 25

V3 30

V4 29

MON 862 V2 55

V3 45

V4 38

Taken from MRID 486367

- 01

Table 2 Western Corn Ro otworm Larval Recovery 10 Days a fter Infestation on to Seed Mats Generated with

MON 862 and Non

- Bt seed Population % Recovered from MON 862 % Recovered from Non

- Bt Crop Characteristics (CC) susceptible colony 0.33 ±0.33 12 ±9.39 Brookings USDA susceptible colony 0.50 ±0.50 26 ±10.9 Columbia USDA resistant colony 11 ± 2.86 13.66 ±3.63
CC and Brookings are susceptible colonies; USDA resistant colony supplied by Hibbard Lab.

Taken from MRID 486367

- 018 Table 3 Age Structure of Western Corn Rootworm on MON 862 and Non

- Bt Seedling Mats Population % Mean First Instar % Mean Second Instar % Mean Third Instar

CC Susceptible on non

- Bt 0 0 100

CC Susceptible on MON 862 0 100 0
Brookings on non

- Bt 0 4.2 95.8 Brookings on MON 862 0 100 0

Resistant USDA on non

- Bt 0 8.9 91.1

Resistant USDA on MON 862 0 35 65

Modified from MRID 486367

- 01

Figure 1 Western Corn Rootworm Recovery 10 Days after Infestation with 2 Susceptible Colonies and 1 Resistant Colony on MON863 Seedling Mats (MRID 486367

- 01)9 Figure 2 Age Structure of Western Corn Rootworm larvae Recovered on MON 862 and Non

- Bt Seedling Mats (MRID 486367

- 01)

c. Monsanto 8/31/2012 submission :

EPA received update s on the progress of developing a proactive resistance monitoring program for northern corn rootworm ( Diabrotica barberi ). ABSTC held a workshop with expert entomologists in November 2012 at the Entomological Society of America meeting to discuss obs tacles associated with northern corn rootworm field collections, rearing, and diet bioassays. The group concluded that current technical and logistic constraints make it impossible to reliably monitor for northern corn rootworm. There are no currently avai lable established techniques for collecting and rearing of this rootworm species. An action plan was proposed to address the present constraints and should be available by 2013.

d. Monsanto 9/28/ 2012 submission :

Monsanto summarized the conclusions of the prior two SSA updates and their efforts to establish a single

on

- plant assay. In their SSA

- update, the registrant reported that the first seed increase for MON 862 was successful but would take an additional 1

- 2 generations to generate sufficient amounts o f seed for further experiments. Monsanto expect ed that by October 2012 sufficient seed should be available to continue

their work with SSAs and Cry3Bb1 expressing seedlings

.

Monsanto also investigated the feasibility of single on

- plant assays as a diagno stic tool for Cry3Bb1 resistance detection. The methodology was tested on six populations collected from either high

- pressure fields or from performance inquiry sites in 2011; the Bt seeds utilized in the on

- plant assay were from

MON 88017 stock. On

- plant results were compared to diet bioassay results. Both assays were designed to

evaluate western corn rootworm survival and growth inhibition. Survival in the diet bioassay was assessed10 as percent mortality and in the on

- plant assay as percent recovery. Growt h inhibition was assessed as average weight of survivors in diet bioassays and as head capsule width and dry weight for larvae that had

recovered after the on

- plant assay exposure time.

Monsanto ’s Results and Conclusions: Diet bioassays: the registrant stated that a verage percent mortality and/or avera ge weight of survivors indicated that all populations were less susceptible than expected based on the historical baseline data (except Niebur, CO) (see Table 4 )

.

On

- plant assay: Monsanto reported that on

- plant r esults indicated that Niebur (CO) and Humphrey (NE) populations remained susceptible to Cry3Bb1 . The remain ing four populations , however, had lower susceptibilities based on their limit ed response to MON 88017 expressing plants (see Table 4 )

.

Monsanto concluded diet bioassay and on plant

- assay results were generally comparable with one exception (Humphrey population, NE). Monsanto continue d to develop, reconcile, and optimize their assay systems for use in corn rootworm r esistance monitoring. Additional field populations would be

tested side

- by

- side using both assays. The results would be compared with results of mechanistic assays (assays of resistance mechanisms) to determine which assay best represents the susceptible s tatus of a rootworm population.

Table 4 Diet Bioa s say and Sublethal Seedling Assay Results 1 for CRW from High

- Pressure and/or Performance Inquiry Fields (2011 Season)

Location Diet B ioassay R esults Plant B ioassay (Control compared to MON 88017)

EC 50 2

(95% CL) Diagnostic

Concentration (170.8 μg/cm 2 ) % Larval Recovery Ave Head Caps. Width Ave Dry Weight

Avg. %

mort. Avg.Wt

per Surviv. C ontrol MON

88017 P

Value control MON

88017 P

Value control MON

88017 P

Value

Niebur Field (CO) 24.99 (14.4

- 35.2) 46 0.16 23 6 .012 0.48 0.38 .0002 0.77 0.24 0 .0002

Onslow (IA) * 43 0.32 9 12 .710 0.48 0.42 .004 0.73 0.39 0 .004

Shell Rock (IA) * 29 0.36 17 11 .352 0.51 0.47 .009 0.98 0.59 0 .002

Mankato

(MN) * 51 0.37 33 23 .135 0.51 0.39 .001 0.81 0.60 0 .002

Humphrey (NE) * 22 0.30 29 8 .002 0.52 0.35 < 0 .05 0.86 0.19 < 0 .05

Trent (SD) * 19 0.27 26 24 .836 0.47 0.43 0 .274 0.67 0.46 0 .314

Historical

Range 3 7.3

- 119.6 (2.1

- 153.7) 24

- 89 0.01

- 0.13 - - - - - - - - -

Modified from Monsanto submission dated 9/28/2012 (p. 3) *Data failed to converge: EC 50 could not be determined

1 Diet bioassay and plant

- based bioassay results from Monsanto report: 2011 western corn rootworm resistance monitoring for Cry3Bb1

2 EC= Effective Concentration; CL Confidence Limit

3 Historical = Ranges from routine monitoring results from 2007

- 201011 III. MONSANTO’S 201 1 WESTERN CORN ROOTWORM RESISTANCE MONITORING

DATA FOR CRY3Bb1 (DIET BIOASSAYS) (MRID 489283

- 01)

Monsanto submitted the 2011 Cry3Bb1 resistance monitoring report containing diet bioassay results for the routine annual monitoring program as well as follow up investigations for fields with unexpected Cry3Bb1 damage in 2010 or 2011. In addition to conduc ting diet bioassays, the registrant also conducted

on

- plant assays on a subset of the populations to pro vide a more in

- depth assessment of populations in question . Monsanto also submitted a summary of the 2011 Problem Investigations (PI) and their Best

Man agement Practices (BMPs) implemented in those fields during the 2012 corn growing season

.

a. Summary of t he 2011 random population sampling results

DM Crop Research Group and Monsanto Company made corn rootworm collections throughout the US

Corn Belt in 2011 (on behalf of ABSTC)

. T hirteen geographically distinct populations of WCRW were collected from Nebraska, Iowa, and Illinois where Cry3Bb1 adoption and western corn rootworm

infestations have been historically high

. All insects were sent to Custom BioProdu cts and maintained in the lab for egg collections; eggs were maintained in a diapausing condition until initiation of bioassays in

March 2012. Bioassay tests were conducted on the progeny of the 12 popul ations with typically four to 10 replicates to test t he susceptibility of corn rootworm to the Cry3Bbl protein ( Table 5 ). A non

- diapausing laboratory population maintained by Monsanto produced enough neonates for four replications. In an effort to improve bioassays a new lot of protei n was developed with a concentration of 9.9 mg/ml. This new lot of protein allowed for the diagnostic (highest) concentration to increase from 170 μg/cm 2 to 341.6 μg/cm 2 . Both concentrations were used to determine percent mortality and average weight per p opulation (see Table 6 ) . Further methodology descriptions were provided in Appendix 1 of MRID 489283

- 01 and

will not be discussed here

.

Monsanto concluded the 2011 diet bioassay results suggested that four of the 13 populations obtained from high Bt adoption areas had reduced Cry3Bbl susceptibility r elative to the historical range and other 2011 populations tested . T hose populations with reduced Cry3Bb1 susceptibility were from F loyd (IA)

, Hamilton (IA)

, Buchanon ( I A )

, and Stant on ( NE) counties (see Table 6 )

. These r outine m onitoring

collections were intended to come from non

- Bt expressing corn or other non

- corn host plants in areas of high CRW

- trait adoption in Illinois, Iowa, and Nebraska. Since the corn plants from which the beetles were collected in 2011 were not specific ally identified (gene

- checked), Monsanto further investigated

. The y determined that the populations with reduced susceptibility for Cry3Bb1 were obtained from Cry3Bb1 expressing corn fields. It was , t herefore, concluded that the reduced susceptibility to C ry3Bbl observed in these populations wa s not unexpected and not representative of populations collected from

non

- Bt fields. Going forward these four fields will be treated as Performance Inquiry (PI) fields.

Monsanto collected western corn rootworm from th ese locations in 2012. For 2013, one of the BMPs identified by Monsanto for PI fields will be implemented for each of these fields. In addition, since the

r outine m onitoring collections for 2012 were already complete Monsanto was also gathering similar

pro duct information for fields where beetles were collected from in 2012. The process for making these collections will be amended through ABSTC to include identification of the corn product in the relevant

field for collections in 2013.

Monsanto reported th at t here were six populations (McLean, Champaign and Piatt in IL, Scott and Cerro Gordo in IA, and Sherman, NE) that had EC 50 results that fell within historical ranges and were comparable to the laboratory control colony. Two populations (Story, IA and Yo rk, NE) were below the

historical range, while one population had an elevated EC 50 (Stanton, NE)

. EC 50 s could not be estimated12 for four populations ( Floyd, Hamilton and Buchanon, IA, and Clay, NE) because only one replicate was available for these sites

. Considering survival and larval mass at the previously used diagnostic concentration (170.8 ug/cm 2 ), there were seven populations (Champaign and P iatt (IL), Scott and Story (I A ) , and Clay, Sherman and York ( NE) ) for which the mortality and average larv al w ere within the historical range ( Table 5 ). There were four populations (McLean, IL and Cerro Gordo, Floyd and Hamilton ( IA ) ) for which the mortality was within the historical range ; the weight was , however, higher than the historical range at 170.8 ug/cm 2 . T wo population s (Stanton, NE and Buchanon, LA ) had average mortality and larval weights outside the historical range at this concentration.

Table 5 . Population Mean EC 50 s, Perce ntage Mortality, and Mass of Surviving Larvae at 170 μg/cm 2 from Routine Monitoring in 2011

Population EC 50 1 Response at 170 μg/cm 2 Field History

(95% CI)

μg Range

status % mortality Range

status Ave weight/

survivor Range

status 2011 2012

Mc Lean Co, IL 19.92 (5. 1

- 35.3) Within 64 Within 0.17 Above VT3 2 Rotate

Scott Co, IA 14.45 (5.6

- 21.7) Within 6 9 Within 0.08 Within Non

- Bt Non

- Bt

Champaign Co,

IL 9.72 (2.7

- 16.8) Within 54 Within 0.07 Within Competitor product SSX RIB 2

Piatt Co, IL 15.71 (4.0

- 26.8) Within 61 Within 0.07 Within Competitor product VT3+SAI 2

Floyd Co, IA * N/A 32 Within 0.27 Above VT3 SSX RIB
Hamilton Co, IA * N/A 29 Within 0.32 Above DeKalb 2 VT3

Story Co, IA 1.08 (0.1

- 3.5) Below 49 Within 0.12 Within DeKalb Competitor product Clay Co, NE * N/A 67 Within 0.07 Within Corn 2 Corn 2

Sherman Co, NE 25.97 (17.9

- 33.5) Within 60 Within 0.13 Within VT3 VT3

York Co, NE 5.66 (1.4

- 11.0) Below 87 Within 0.03 Within Unknown Competitor product

Stanton Co, NE 151.99 (4.0

- 26.8) Above 22 Below 0.24 Above VT3 VT3

Cerro Gordo Co,

IA 12.93 (9.5

- 16.4) Within 60 Within 0.19 Above Corn 2 Rotate

Buchanon Co, IA * N/A 20 Below 0.34 Above VT3P 2 VT3

Monsanto Lab colony 10.23 (3.1

- 16.7) N/A 89 N/A 0.11 N/A N/A N/A

Historical Range 3

2007

- 2 010 at 170 μg/cm 2 7.30

- 119.60 (2.1

- 153.7) N/A 24

- 89 N/A 0.01

- 0.13 N/A N/A N/A

Modified from MRID 489283

- 01 ( Table 1

, p. 9 ) *Data failed to converge: EC 50 could not be determined

Green shaded populations were collected from within Cry3Bb1 expressing maize fields.

Pink s haded fields indicate populations that have EC 50 s, average weights above and/or % mortality below the historical range.

1 Effective concentration of 341.6 μg/ml ; 2 VT3= VT Triple Pro ( MON 88017 x MON810); SSX RIB= SmartStax Refuge in the Bag (MON89034 x TC15 07 x MON 88017 x DAS

- 59122

- 7); SAI= Soil applied insecticide; D eKalb product, trait unknown; Corn= Research field

used to tra p CRW beetles, no trait planted, or trait (if any) unknown; VT3P= VT Triple Pro (MON89034 x MON 88017 ); 3 Ranges from

routine monitoring results from 2007

- 201013 Table 6 . Comparison of 2011 Percent M ortal ity at T wo Different H ighest C oncentrations T ested

Location New Diagnostic Concentration (341.6 μg/cm 2 ) Previous Diagnostic Concentration (170

. 8 μg/cm 2 ) % mortality Average weight % mortality Average weight

Mc Lean Co, IL 65 0.07 64 0.17

Scott Co, IA 79 0.01 69 0.08
Champaign Co, IL 68 0.02 54 0.07
Piatt Co, IL 74 0.02 61 0.07 Floyd Co, IA 39 0.14 32 0.27
Hamilton Co, IA 32 0.22 29 0.32
Story Co, IA 67 0.04 49 0.12 Clay Co, NE 100

-- 67 0.07
Sherman Co, NE 58 0.05 60 0.13
York Co, NE 77 0.02 87 0.03

Stanton Co, NE 35 0.15 22 0.24
Cerro Gordo Co, IA 75 0.05 60 0.19
Buchanon Co, IA 22 0.28 20 0.34
Monsanto Lab colony 2011 90 0.12 2 89 0.11 2

BPPD m odified Table from MRID 489283

- 01 , Appendix 1 Green shaded populations were collected from within Cry3Bb1 expressing maize fields.

Pink s haded fields indicate populations that have EC 50 s, average weights above and/or % mortality below the historical range.

1 S usceptible lab control colony is a non

- diapausing strain maintained by Monsanto

2 Weight of susceptible colony on isoline corn Shaded populations identified by BPPD are likely Cry3Bb1 resistant

b. Diet bioa ssays of collections from performance inquiry fields

Monsanto’s objective was to characterize western corn rootworm populations in areas of the Corn Belt

where elevated levels of corn root feeding had occurred. In an effort to improve diet bioassays, a new lot of protein was developed with a concentration of 9.9 mg/ml. This new lot of protein allowed for the highest (diagnostic) bioassay concentration to increase from 170 μg/cm 2 to 341.6 μg/cm 2

. All 2011 diet bioassay results were obtained with this new protein source. In addition, a new diet plate recipe was developed (no additional information was provided regarding th is change in MRID 489283

- 01)

.

Thr ee populations were collected from problem fields in 2011 and eleven fields where high corn

rootworm pressure was previously observed in 2010 but w h ere no insects had been collect ed at the time

. Insects were sent to Custom BioProducts and maintained in the lab for egg collections; eggs were maintained in a diapausing condition until initiation of bioassays in March 2012. Of the total eggs

collec ted from the 14 populations, only 12 populations produced enough eggs to conduct bioassays. A

non

- diapausing laboratory population maintained by Monsanto produced enough neonates for four replications. Further methodology descriptions were provided in Appe ndix 2 of MRID 489283

- 01 and are not discussed here

.

Three populations had mean EC 50 s that fell within the historical range for diet bioassays and were comparable to the results obtained for the laboratory control treatments (Niebur, CO; Prophetstown, IL; and Gayville, SD). One population (Livengood, CO) had a mean EC 50 that fell below and another population’s EC 50 (Schutte, CO) fell above the historical range. For seven populations, the mean EC 50 could not be estimated (Maynard, Shell Rock, and Onslow ( IA ) ; Chatfield and Mankato ( MN ) ; Humphrey, ( NE ); and Trent ( SD) ) (see Table 7 ).14 Table 7 . P opulation M ean EC 50 s, Percent Mortality, and Mass of S urviv ing Larvae at 170 μg/cm 2 from 2011

Problem Fields

BPPD modified Monsanto’s Table 1 (p. 9 of MRID 489283

- 01).

1 Effective concentration of 341.6 μg/ml

2 VT3= VT Triple (MON 88017 x MON810); SSX RIB= SmartStax Refuge in the Bag (MON89034 x TC1507 x MON 88017 x DAS

- 59122

- 7); SAI= Soil applied insecticide; DeKalb product, trait unknown; VT3P= VT Triple Pro (MON89034 x MON 88017);

3 Ranges from routine monitoring res ults from 2007

- 2010

Using survival and body mass at the previously highest concentration tested (170.8 μg/cm 2 ), one population was within the historical range (Livengood, CO) (see Table 8 ) . For eight populations, the average mortality was within the historical range but average weight/survivor was above the historical Populations EC 50 1 Response at 170 μg/cm 2 Field History (95% CI)

μg Range

status Percent mortality Range

status Ave weight/

survivor Range

status 2010 2011 2012

Maynard, IA * N/A 33 Within 0.27 Above High

pressure Rotate

& trials Rotate &

trials

Shell Rock,

IA * N/A 29 Within 0.36 Above High

pressure Rotate

& trials SSX RIB

Humphrey,

NE * N/A 22 Below 0.30 Above High

pressure Rotate

& trials Competitor

Onslow, IA * N/A 43 Within 0.32 Above High

pressure Rotate

& trials VT3 2 /

VT3P 8 +SAI 4

Livengood,

CO 3.30 (1.1

- 6.5) Below 46 Within 0.13 W ithin Unknown CRW

claim

field in

2011 Rotate,

SSX RIB 3

, competitor

Niebur, CO 24.99 (14.5

- 35.2) Within

46 Within

0.16 Above Unknown CRW

claim

field in

2011 VT3 2 /

VT3P+SAI 4

c ompetitor

Schutte, CO 140.67 (95.1

- 246.2) Above 29 Within 0.30 Above Unknown Rotate

& trials Rotate,

SSX RIB

Gayville, SD 28.87 (19.7

- 37.8) Within 43 Within 0.17 Above High

pressure Rotate

& trials Rotate

Trent, SD * N/A 19 Below 0.27 Above High

pressure Rotate

& trials SSX RIB

Chatfield,

MN * N/A 19 Below 0.20 Above High

pressure Rotate

& trials SSX RIB

Mankato, MN * N/A 51 Within 0.37 Above High

pressure Rotate

& trials Rotate

Prophetstown,

IL 50.0 (25.1

- 78.5) Within 58 Within 0.18 Above High

pressure High

press. Rotate &

trials

Amber, IA No results

– not enough insects to run bioassays High

pressure Rotate

& trials VT3/VT3P

+ SAI

Oelwein, IA No results

– not enough insects to run bioassays High

pressure High

press. Rotate &

trials

Monsanto Lab colony 10.23 (3.1

- 16.7) N/A 89 N/A 0.11 N/A N/A N/A N/A

Historical Range 3

2007

- 2010 at 170 μg/cm 2 7.30

- 119.60 (2.1

- 153.7) N/A 24

- 89 N/A 0.01

- 0.13 N/A N/A N/A N/A15 control. Three populations (Humphrey (NE), Trent (SD), and Chatfield (MN)) were above the historical range for percent mortality and average weight/survivor. Monsanto concluded that collectively these results indicated that eight populations (Maynard, She ll Rock, and Onslow (IA), Schutte Fields (CO), Chatfield and Mankato (MN), Humphrey (NE) and Trent (SD)) of the 12 populations obtained from fields with PIs had potentially reduced Cry3Bb1 susceptibility compared to the historical range and other 2011

popu lation samples obtained from routine monitoring efforts.

Table 8 . Comparison of 2011 Percent Mortality of PIs at Two Different Effective Concentrations

Location Effective Concentration (341.6 μg/cm 2 ) Effective Concentration (170.8 μg/cm 2 ) % mortality Average weight % mortality Average weight
Maynard, IA 50 0.24 33 0.27
Shell Rock, IA 42 0.34 29 0.36 Humphrey, NE 25 0.36 22 0.30
Onslow, IA 39 0.37 43 0.32 Livengood, CO 40 0.12 46 0.13
Niebur, CO 58 0.17 46 0.16
Schutte, CO 44 0.27 29 0.30 Gayville, SD 53 0.17 43 0.17

Trent, SD 15 0.29 19 0.27
Chatfield, MN 19 0.28 19 0.20
Mankato, MN 54 0.31 51 0.37 Prophetstown, IL 64 0.23 58 0.18
Monsanto Lab Colony 1 2011 90 0.12 2 89 0.11 2

Modified from MRID 489283

- 01 ( Appendix1)

Pink s haded populations identified by Monsanto as populations with reduced Cry3Bb1 susceptibility

. 1 Susceptible lab control colony is a non

- diapausing strain maintained by Monsanto

2 Weight of susceptible colony on isoline corn

c. On

- Plant assay results from high pressure areas or problem field s identified in 2010

Six of t he populations collected in 2011 came from 2010 high pressure or performance inquir y fields. These populations were also tested with on

- plant assays at a Monsanto facility (Chesterfield, MO)

. A single seed of MON 88017 or non

- Bt expressing corn variety was planted into a three liter pot (between

3

- 10 pots (or replicates) per population) and maintained under conditions favorable for growing corn. At

the V4

- V5 stage of the plant, infestations were made with 30 neonates/plant.

Two of the populations remained highly susceptible to Cry3Bb1 (see Table 9 ) as judged by the relative survivorship between MON 88017 and non

- Bt expressing plants (Humphrey, NE; Niebur, CO). The remaining four populations had reduced susceptibility on MON 88017 plants (Shell Rock and Onslow, IA; Trent, SD; and Mankato, MN) but lower larval survival on Bt than on non

- isoline plants (except for Onslow population). In addition to lower surv ival, all populations had smaller head capsule width and lower larval weights (except for Trent p opulation) than larvae from iso line plants. If the four populations with reduced susceptibility exhibited resistance, then it appeared to be incomplete resistance in all cases.

Monsanto concluded that these results were generally in agreement with the results obtained from diet bioassay s (excep t for the Humphrey , NE population ).16 Table 9 . On

- Plant Assay s Conducted in 201 1 with Populations from High Pressure or PI Fields (2010 Data)

Population % Larval Survival Average Head Width (mm) Average Dry Weight (mg)

Isoline Cry3Bb1 P

- value Isoline Cry3Bb1 P

- value Isoline Cry3Bb1 P

- value Shell Rock, IA 17 11 0.352 0.51 0.47 0.009 0.98 0.59 0.002 Humphrey, NE 29 8 0.002 0.52 0.35 <0.05 0.86 0.19 <0.05
Onslow, IA 9 12 0.710 0.48 0.42 0.004 0.73 0.39 0.004

Niebur field, CO 23 6 0.012 0.48 0.38 0.0002 0.77 0.24 0.0002
Trent, SD 26 24 0.836 0.47 0.43 0.274 0.67 0.46 0.314
Mankato, MN 33 23 0.135 0.51 0.39 0.001 0.81 0.60 0.002
Waterman lab colony 14 * 0 0.027 0.39

-- N/A 0.42

-- N/A

Table has been modified from Monsanto submission MRID 489283

- 01(Tables 2

- 4, p. 41) Red shading indicates suspected resistant populations; green shading indicates resistant population (BBPD determination)

. * BPPD notes low survival of reference strain on isoline corn. No explanation wa s provided in the submission.

d. I mplementation and performance of Monsanto’s CRW Best Management Practices (BMPs) in

2011

The majority of PIs in 2011 occurred in Colorado, Illinois, Iowa, Kansas, Nebraska, and Minnesota. Monsanto reported that a s a conservative response to the increased number of PIs r eported , a program of

BMPs was implemented in these PI fields. Some PIs were also reported in Michigan, Montana, Oklahoma, and South Dakota. The BMPs were recommended to growers in the following order :

1. Rotation of the affected field to a non

- host crop such as soybe ans, which breaks the CRW cycle;

2. Use of Genuity SmartStax® corn hybrids on PI field because of dual CRW modes

- of

- action pyramid and high level of CRW control;

3. If rotation or switching to Genuity SmartStax corn are not an option for growers, use of

soil

- or foliar

- applied insecticides with MON810 x MON 88017 (VT3) and MON89034 x MON

88017 VT3P) to manage larv ae and adult populations of CRW; or 4. Growers also have a choice of planting Monsanto ’s MON89034 L epidopter an protected PIP product, a competitor’s CRW protected PIP products or non

- Bt corn with soil and foliar applied

insecticide.

Monsanto followed up three times with every grower who experienced CRW PIs in 2011 to ensure that they under stood, chose

, and implemented the right BMPs. Monsanto surveyed all PI fields in 2012 to verify that the BMPs were implemented but also to assess the CRW damage during the following growing

season. Figure 3 below identifies the percentage of growers who ch ose BMPs 1

- 4 in the five states

identified with the greatest amount of PIs.

Corn rootworm collections from 2010/2011 PI fields were obtained during the 2011 season, and best management practices were implemented (as described above) during the 2011 and/or 2012 season (see

Table 7 for field history). Of the 2010 PI fields that were rotated to soybean in 2011

, a small set of fields were used for a replicated test of the BMPs, which provided the insect source for 2011 collections. In 2012, these PI fields were once more subjected to BMPs or test plots. The three PI fields from 2011 had beetles collected, were pl anted to corn again with appropriate BMPs during 2012 , and surveyed the following year for root damage. All sites had effectively implemented some BMP strategy during 201217 and had therefore reduced CRW densities and controlled any putative Cry3Bb1 resistan ce at those

locations

.

Figure 3 . Distribution of BMPs by State: CO = Colorado, IA = Iowa, IL= Illinois, KS = Kansas, MN = Minnesota, NE = Nebraska, and SD = South Dakota ( Figure taken from MRID 489283

- 01)

IV. BPPD IRM TEAM REVIEW OF MONSANTO’S SUBMISSIONS AND OVERALL

CONCLUSION S

a. 2011 Sublethal Seedling Assay

The 2011 Sublethal Seedling Assays update (see Section II) demonstrates that Monsanto has made substantial progress towards developing a Cry3Bb1 seedling assay as a functional bioassay for resistance

detection (MRIDs 484368

- 01 and 486367

- 01)

. Based on the protein expression data of MON 88017 and

MON 862 seedlings, BPPD concurs that MON 862 is a better choice for sublethal seedling assays and

should provide greater sep aration between susceptible and potentially resistant larvae. Based on the data submitted, BPPD agrees with the registrant that a comparison between the distribution s of instar development should also provide greater power than a comparison between surviva l on Bt and non

- Bt seedling mats

. BPPD questions

, however, whether the distributions of the lab and field population s on Bt seedling mats need to be completely non

- overlapping before a field population is called resistant. In particular, BPPD is concerned with possible cases of incomplete resistance (visible on Bt corn only) and cost to resistance (visible on no

- Bt corn only) that may delay development and cause partial overlap of the two distributions (control and field colony). Cost to r esistance in western corn rootworm has so far been

identified with respect to fecundity for laboratory selections of field

- derived populations ( Oswald et al.18 2012 and Meihls et al. 2012 )

. If statistical criteria for resistance determinations are too string ent , BPPD is

concerned that this would limit the effectiveness of the resistance monitoring program to detect resistance

.
Resistance evolution in the field is likely to select for different resistance genes tha n artificial selection experiments carried out i n the laboratory (SAP 2009)

. BPPD agrees with Monsanto that it will be important to test field resistant populations from Dr. Gassmann’s lab with the registrant’s SSA method to

see whether the results of the U SDA resistant colony can be replicated. Since the re are still uncertainties regarding mechanisms and typ e of resistance for corn rootworm on Bt toxins (though it is suspected that resistance may be incomplete (Gassmann et al. 2011) ), BPPD proposes to test for “ a significant difference in age distributions ” when conclud ing whether a population is resistan t rather than requiring that

“ distributions are non

- overlapping ”

. BPPD also proposes that the registrant use a second measure (i.e., body mass) to confirm resistance. In other words, two independent measures taken together would be

needed to identify resistant corn rootworm populations. Once the SSA methodology has been fully worked out by Monsanto, it will be important to develop and implement a statistical approach that proactively identif ies resistant populations.

b. 2011 On

- Plant Greenhouse Assay

In 2012, Monsanto investigated the feasibility of an on

- plant assay using MON 88017 seedlings for 2011 problem field insect collections and insect populations from high pressure areas

, while also using diet bioassays as a comparison. BPPD reviewed the preliminary results submitted to the Agency (Letter dated

9

- 28

- 2012 ) and was unclear as to the distinction between a Cry3Bb1 problem field and a Cry3Bb1 field

tha t experienced unusually high insect pressure. Typically one would expect both observations to occur in fields with heavy damage

. The registrant chose to measure decreased Cry3Bb1 susceptibility with

“ percent recovery ” for on

- plant assays and “ percent morta lity ” for diet bioassay s

. BPPD recommends that

Monsanto use percent mortality for on

- plant assays because data have shown that percent recovery was

not reliable at identifying putative resistan t individuals. Cry3Bb1, as are all currently registered corn

ro otworm toxins, is non

- high dose, which may explain why even susceptible neonates could rec over from toxic exposure to the toxin. Aspects of the on

- plant methodology were unclear, such as the assay duration.

BPPD recommends that Monsanto consider using a 17

- day period

, as was done by Gassmann’s lab

, since

this time period allow s for insects to reach third instar development

. Head capsule width was another

measure used in the on

- plant assays. BPPD notes that four out of six field popul ations were identified to
have (statistically significant) smaller average head capsule width s compared to the susceptible lab

colonies. Hence “ head capsule width ” as well as “dry weight” appear to show greater promise in separating susceptible from resistant populations

. BPPD recommends that Monsanto also use MON 862 plants for on

- plant assays since the greater toxin expression in this Cry3Bb1 seed may provide greater
sensitive to separate resistant from susceptible individuals

. P ercent mortality, average dry weight, and /or

hea d capsule measurements are recommended for the on

- plant assays

.

c. 2011 Diet and On

- Plant Assays

In their 2011 annual monitoring report (MRID 489283

- 01) , Monsanto tested a new highest concentration (341.6 μg/cm 2 ) with a new protein source in addition to 170 .8 μg/cm 2

, the maximum concentration used in previous monitoring assays

. Because of the switch in protein source, a direct comparison should

, therefore

, not be made between the 2011 diet bioassay results and the 2007

- 2010 results (listed in the

same tab le)

. Changes in toxin sources have b een a periodic challenge for diet bioassays and resistance monitoring. In such situations, the comparisons could only be made between results of control and field19 populations within a specific year. Figure 4 shows that the percent mortality and average weight of surviving insects of a population were more strongly correlated when 341.6 μg/cm 2 was used instead of

the previously established 170.8 μg/cm 2

. The variance was gre ater at the lower concentration; the R 2 at the (new) highest concentration was 0.9089 compared to the R 2 of 0.7685 at 170 μg/cm 2

. T his suggests that the higher concentration likely provide d a better estimate of susceptibility for two measures (mortality and weight of survivors). BPPD included all field populations from the annual routine monitoring report ( Tabl e 6 ) in the correlations shown in Figure 4 (excluding Clay Co, NE because all

individuals tested died )

. BPPD excluded the results of the non

- diapausing la boratory colony because it

was unclear how representative these were of truly susceptible western corn rootworm field populations. BPPD notes, h owever, that even with the new higher concentration

, diet bioassay results were still

variable . For example, two populations had lower percent mortality at 341.6 μg/cm 2 than at 170.8 μg/cm 2

(York and Sh erman, NE)

.

A lthough Monsanto implemented BMPs in 2012 on four fields identified with potentially resistant corn rootworm populations ( Floyd Co (IA), Hamilton Co (IA), Buchanon Co (IA), and Stanton Co (NE) )

, BPPD recommends that the registrant monitor the se sites in 2013 . These four corn rootworm populations

where collected from Bt rather than non

- Bt crops (or alternate hosts) during the annual monitoring

activities and had a low er percent mortality (range 22%

- 39%) and higher average weight (range 0.14 mg

- 0.28 mg) than the laboratory control populations ( see Table 5 and Table 6 ). Also, Stanton (NE) had a much higher mean EC 50 (151.99 μg/cm 2 ) compared to the lab colony (10.23 μg/cm 2 ). T hese results suggest that these four populations may be resistant or in the process of developing resistance

.

BPPD notes that the registrant was unable to obtain EC 50 s with the new protein source for seven out of 14 populations. This is further evidence that the current diet bioassay methodology does not provide a functional resistance monitoring tool for corn rootworm.

Furthermore, when body weight results are obtained with Bt diet bioassay for the purpose of determining whether a corn rootworm population is resistant, BPPD recommends that the comparisons be made

between the tested field population and a resistant reference colony rather than the susceptible control colony. More information can be extracted from the first comparison.20 Figure 4 . Correlation between percent mortality and average weight of survivors in a particular populatio n
at two highest concentration s tested (BPPD generated graph using Monsanto’s data from Table 6 of this

review )

Monsanto used their improved diet bioassay methodology for 2011 populations that originated from unexpected damage sites as well as high

- press ure areas identified in 2010. Monsanto further followed up

on six of the PI s with their single on

- plant assay and measured percent mortality, head capsul e width, and average dry weight for each population raised on Bt and non

- Bt expressing corn plants. The statistical analysis was done for the field population s on the two treatments. T he registrant reported that four populations were of concern ( greater survival on Bt but smaller head capsule width and average weight)

and could exhibit incomplete resistance

. Those populations were : Shell Rock ( Butler Co, IA), Onslow ( Jones Co, IA), Mankato ( Blue Earth Co, MN), and Trent ( Moody Co, SD). BPPD identified one population where at least two measures from the on

- plant assay indicated that there was no significant

d ifference between treatments for determining potentially resistant populations and concludes that the Trent, Moody Co (SD) population is likely resistant to Cry3Bb1

. This population had no significant

difference in % survival, average head capsule width, a nd average dry weight on Bt and non

- Bt plants.

T he remaining three populations (Shell Rock, Onslow, and Mankato) had no significant difference in survival on Bt and isoline corn, while average head width and body weight differed significantly. These

result s together are inconclusive

, and it cannot be concluded that the other three populations are resistant

. BPPD recommends , however, that Monsanto monitoring these four locations for further field failures in the coming future

. R2 = 0.9089 R2 = 0.7685

- 0.05 0 0.05

0.1 0.15

0.2 0.25

0.3 0.35

0.4

0 20 40 60 80 100Ave weight of survivors

Percent mortality

341.6 ug/cm2 170.8 ug/cm221 F igure 5

. 2013 U pdated map showing the general areas in the US Corn Belt that have had Cry3Bb1 fields with heavy damage and/or resistant western corn rootworm populations (2009

- 2012) 

### Kansas

Oklahoma Colorado Nebraska South Dakota Minnesota

Wisconsin

Iowa

Illinois22 d. Modifications to the Cry3Bb1 Resistance Assessment Procedures

In July, 2013, Monsanto’s Cry3Bb1 registrations were amended to include a new resistance d etection

strategy. This revised approach incorporated field damage triggers for unexpected crop damage as well as

the use of on

- plant bioassays to determine resistance. Major aspects of this approach include the following (paraphrased from the terms and conditions of registrations 524

- 552, 524

- 576, and 524

- 606):

• Monsanto is required to investigate “performance inquiries” (i.e., reports of unexpected corn rootworm damage to Cry3Bb1 corn) from growers. Fields with unexpected damage that are

confirmed to be Cry3Bb1

- expressing and experienced root damage with a Node Injury Score (NIS) > 1.0 on at least 50% of plants sampled are subjected to further investigation.

• CRW are collected from the field in question (at least 250, ideally 500 or more). Collections ma y be extended to any bordering cornfields, if necessary to obtain sufficient CRW adult individuals. If sampling is unsuccessful, Monsanto will visit the farm or field the following year to reattempt collections (provided the grower is still a Monsanto cust omer). Collected populations are investigated for potential resistance using an on

- plant bioassay.

• Monsanto works with the affected grower to implement CRW management practices. These options include (but are not limited to): rotation to non

- host crop ( e.g., soybean), use of pyramided products, use of additional CRW control tools (e.g., soil insecticides, seed

- applied insecticides, chemigation),

and use of alternate CRW PIPs.

• A population is considered resistant if the following criteria are met:

1. An init ial performance inquiry investigation exceeds the root damage threshold and Cry3Bb1 protein levels are found to be within the documented range for that hybrid; and

2. Single, on

- plant bioassays of insect collections from the affected fields show the following : i. A statistically significant difference in measures of either lethality/mortality or sublethal effects (growth/development) between the field population and the control population on Cry3Bb1 corn and

ii. A lack of a statistically significant difference in the same measures of the field population raised on Bt corn and non

- Bt corn plants.

• In the event the on

- plant bioassays are inconclusive (e.g., the results of the statistical analysis are unclear because of low sample sizes) or another reasonable explanation for the unexpected damage exists (e.g., high pest pressure and/or high plant stress) another year of CRW adult collections and additional testing will be conducted to determine resistance.

These monitoring procedures were not in place when Monsanto conduc ted testing of the 2011 CRW populations (tests were performed in 2012). Monsanto, however, did conduct on

- plant assays of six populations collected from performance inquiry fields during 2011 (see Table 9). Four of these populations appeared to meet the second criterion for resistance in that the response to one measure (% larval survival) was statistically similar on Bt and non

- Bt corn plants. One population (Trent, SD) had no significant different responses on Bt and non

- Bt for all three measures (% la rval survival, average head width, and average dry weight). On the other hand, it could not be determined from the report whether the % larval

survival values of the field populations were statistically different from the control population (the first

res istance criterion).23 e. Monsanto’s Best Management Practices

Monsanto reported that they implemented BMPs in all Cry3Bb1 corn fields in 2011 that had experienced unusually high root damage and that were reported to the company as PIs. BPPD agrees with Monsanto ’s approach to take immediate action before a resistance situation is confirmed. BPPD further agrees that crop rotation in problem fields is the best solution to unexpected Bt failure but understands that not every farmer can resort to this type of management practice for logistical reasons . Crop rotation with a non

- corn rootworm host plant should extirpate the offspring of a putative resistant corn rootworm population. BPPD

, however

, cautions that these problem fields do not occur in a vacuum but r ather are planted in a landscape of continuous corn. Any management practice will only alleviate the corn rootworm problem for a short period

of time if resistance has spread outside the managed field . Because of this, it would be more effective to impleme nt BMPs on a wider geographic scale (if possible). Re

- colonization of adult corn rootworm from the continuous corn landscape will spread putative resistance genes back into the managed fields once corn is grown there again . BPPD understands the limitation s face d when implementing BMPs and that it is not possible for Monsanto to require that non

- Monsanto customers ( i.e ., neighboring growers of a managed field) follow their best management practices.

Using SmartStax as the second management practice to Cry3B b1 unexpected damage, as proposed by Monsanto, could be a good option but will increase selection pressure on the second toxin (Cry34/35) with the current 5% refuge requirement. In areas with resistance to Cry3Bb1, SmartStax is essentially a single

trait P IP with a 5% refuge that was designed for an effective two

- toxin pyramid

. The better scientific strategy would be to deploy the pyramid containing the compromised toxin with a 10% refuge.

Monsanto’s third option to apply SAI and adulticides will reduce th e susceptible and resistant individuals proportionally. This BMP will suppress the population density but is not expected to mitigate resistance

unless the field is the ‘remedial action area’ and the population is extirpated

. If corn rootworm populations rebound, managed problem fields will likely fail again if they are planted to Cry3Bb1.

BPPD concludes that while Monsanto’s BMPs are a reasonable mitigatory approach to dea ling with unexpected damage in Bt corn fields, most of the BMPs are not a long

- term solution for managing Cry3Bb1

resistance and/or putative resistance in corn rootworm (except crop rotation or switching to another Bt trait ). Furthermore, if Cry3Bb1 resistance is known to have evolved in a particular populatio n, continued planting of the compromised toxin in that area is not recommended.

f

. Northern Corn Rootworm

Monsanto submitted an update on the resistance monitoring program for northern corn rootworm (NCRW)

. The BPPD IRM team recognizes that NCRW prese nts significant logistical challenges for resistance monitoring including difficulties with sampling, rearing, egg production, and testing with bioassays. For this reason, the BPPD IRM team concurs with ABSTC’s conclusions that at present time, it is not p ossible to effectively monitor NCRW for changes in toxin susceptibility. Despite the technical hurdles presented by NCRW, BPPD continues to recommend that Bt corn registrants work to develop a functional resistance monitoring program for this insect. BPPD notes that one NCRW population was tested with the Sublethal Seedling Assay approach by another registrant with a different corn rootworm toxin (BPPD 2013) and that the assay may hold promise as a practical resistance detection tool. The greater challenge with NCRW, however, appears to be in sampling and laboratory rearing. ABSTC attempted to make multiple NCRW24 collections during 2009

- 2011, but of the total 24 identified locations only 10 resulted in successful collections. Of those 10 populations, only on e produced enough eggs for testing. The BPPD IRM team recommends that ABSTC continue the efforts to collect NCRW from the representative regions of the Corn Belt. Any successful collections should be tested by a suitable bioassay (on

- plant) but can also be used to refine and improve NCRW re aring techniques in the lab. BPPD also recommends that any progress towards developing a resistance monitoring program be reported to the EPA on an annual basis

.

V. ACADEMIC REPORTS OF CROSS

- RESISTANCE BETWEEN CRY3Bb1 AND mCRY3A

At the 2012 Entomological Society of America meeting in Tennessee, th ree independent research teams presented data (currently unpublished) in dicating potential cross resistance between mCry3A and Cry3Bb1. For example, Dr. Aaron Gassmann (Iowa State University) exposed 17 different Cry3Bb1 field resistant colonies to mCry3A and Cry3Bb1 expressing plants and subsequently compared the observed (corrected) survival of each population to both toxins. When plotting the survival on mCry3A against the survival on Cry3Bb1 plants for all 17 p opulations, a positive and strong correlation emerged (P

- value < 0.0001) and lead Gassmann to conclude that Cry3Bb1 field resistant western corn rootworm populations exhibited cross resistance to mCry3A (Gassmann et al. 2012). Dr. Lance Meinke (University of Nebraska

- Lincoln) reported that the Wayne Coun ty western corn rootworm population inflicted comparable root damage and beetle emergence to both Cry3Bb1 and mCry3A field plots (Meinke et al. 2012). Sarah Zukoff (University of Missouri) conducted a labora tory selection experiment in which CRW selected for resistance on Cry3Bb1 corn were exposed to mCry3A in greenhouse seedling assays. These larvae were found to also be tolerant of mCry3A, though not in all populations (Zukoff and Hibbard 2012). While thes e reports are preliminary and have not been subjected to peer review or publication, they are strongly suggestive that cross

- resistance between Cry3Bb1 and mCry3A may occur in resistant CRW populations. The mechanism of resistance in

CRW to Bt toxins is no t well understood, so it is unclear whether cross

- resistance is due to a common physiological trait or other means. Additional data will be needed to fully assess potential cross r esistance and implications for insect resistance management.

BPPD is concer ned with these preliminary reports of cross

- resistance between Cry3Bb1 and mCry3A and the planting of CRW pyramid ed corn expressing Cry3

- Bt toxins in areas with Cry3Bb1 resistant corn rootworm populations. Such pyramids are effectively reduced to a single toxin product deployed with a much lower refuge (5%). There are currently four registered corn rootworm toxins

, t wo of which (mCry3A and Cry3Bb1) would be ineffective in corn rootworm resistant areas . T he selection pressure in these regions

on the remainin g toxi n, Cry34/35, will be tremendous if deployed in a pyramid with Cry3Bb1 and a reduced refuge of 5%.25 VI. CITATIONS

BPPD. 2013. Review of corn rootworm resistance monitoring submissions and data (2009

- 2011) for Bt corn products containing Cry34/35Ab1. Memo from A. Reynolds to A. Sibold, dated March 20, 2013

BPPD. 2012. BPPD IRM Team r eview of Monsanto’s 20 10 corn r ootworm monitoring data, unexpected damage reports for Cry3Bb1 expressing Bt Corn and academic reports of Cry3Bb1 field failures as well as corn rootworm resistance. Memo from JC Martinez to A. Sibold, dated October 11, 2012.

BPPD. 2011. Updated BPPD IRM review of reports of unexpected Cry3Bb1 damage, Monsanto’s 2009 corn rootworm monitoring report, and revised corn rootworm resistance monito ring plan for MON 88017, MON 88017xMON 810, MON 863, MON 863xMON 810, MON 89034xTC1507xMON 88017xDAS

- 59122

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and MON89034x MON 88017

. Memo from JC Martinez to M Mendelsohn, dated Nov 22, 2011.

BPPD. 2010a. Science assessment of Pioneer’s modeling addendum in support of their application of registration for Optimum AcreMax1 Insect Protection. Memo from JC Martinez to M. Mendelsohn, dated February 4, 2010.

BPPD. 2010 b . Review of Monsanto’s 2008 corn rootworm monitoring report and revised corn rootworm

resis tance monitoring plan for MON 88017, MON 88017xMON 810, MON 863xMON 810, MON 89034xTC1507xMON 88017, and MON89034x MON 88017 . Memo from JC Martinez to M Mendelsohn, dated Jun 30, 2010.

BPPD. 2010c . Terms and conditions for Bt corn registrations. September 30, 2010. http://www.epa.gov/oppbppd1/biopesticides/pips/bt

- corn

- terms

- conditions.pdf

BPPD. 2009. Review of Monsanto’s corn rootworm monitoring reports from 2005

- 20 07 for susceptibility to Cry3Bb1 and revised corn rootworm resistance monitoring plan for MON 88017, MON 88017xMON810, and MON 89034xMON 88017. Memo from JC Martinez to M Mendelsohn, dated August 24, 2009.

EPA Scientific Advisory Panel meeting 2009. Trans mittal of meeting minutes of the FIFRA Scientific Advisory Panel meeting held February 23

- 24, 2009 on the evaluation of the resistance risks from using a seed mix refuge with Pioneer’s Optimum AcreMax1 corn rootworm

- protected corn. Memorandum from J.E.

Bai ley (DFO) to Dr. D. Edwards (Director of OPP) on May 6, 2009.

Gassmann A. 2012. Western corn rootworm and Bt maize: a case study illustrating the need for IPM and IRM. Presentation at the ESA in Knoxville, Tennesse.

Head G and PA Price. 2011 Western corn rootworm resistance monitoring for Cry3Bb1. Unpublished report submitted to EPA, MRID 489283

- 01

Head G and N Storer. 2008. Insect resistance management plan for MON 89034 x TC1507 x MON 88017 x

DAS

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Jenkins D. 2012 b . September 2012 update on the conditions of registration for MON 88017, MON 88017 x MON 810, MON 89034 x MON 88017, MON 89034 x TC1507 x MON 88017 x DAS

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89034 x TC1507 x MON 88017 x DAS

- 59122

- 7 RIB Complete, an d MON 89034 x MON 88017 RIB Complete, Monsanto Company is submitting a progress report on the development of a sub

- lethal seedling26 assay (SSA). Letter from Monsanto to Kimberly Nesci (Acting Chief of MPB/BPPD) , dated September 28,

2012 b

.

Jenkins D. 2012. Extension of the condition of registration for developing a northern corn rootworm resistance monitoring program: conditions of registration for MON 88017 , MON 88017 x MON 810, MON 89034 x MON 88017, MON 89034 x TC1507 x MON 88017 x DAS

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MON 88017 x DAS

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- lethal seedling assay (SSA). Letter from Monsanto to Kimberly Nesci (Acting Chief of MPB/B PPD), dated August 31 , 2012 a

.

Meinke, L, T Hunt, G Kruger, R Wright, D Wangila, and K Miwa, 2012. Evaluation of western corn rootworm susceptibility to rootworm Bt traits in Nebraska. Presentation at the Entomological Society of America Annual Meeting. November 13, 2012.

Meihls LN, ML Higdon, MR Ellersieck, BE Tabashnik, and B Hibbard. 2013 . Greenhouse selected resistance to Cry3Bb1

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Moore GC. 2004. Efficacy of MON 880 17 and YieldGard (MON 863) Corn Rootworm Events, Force 3G and Poncho 1250 on Mexican Corn Rootworm, Ellis County, Texas. Texas A&M Cooperative Extension Report.

Nowatzki T, SA Lefko, RR Binning, SD Thompson, TA Spencer, and BD Siegfried. 2008. V alidation of a novel resistance monitoring technique for corn rootworm (Coleoptera: C hrysomelidae) and event DAS

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Oswald KJ, BW French, C Nielson, and M Bagley. 2012. Assessment of fitness cost in Cry3Bb

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